Helper Effect Research Articles

Effects of carbamazepine and metabolites on IL-2, IL-5, IL-6, IL-10 and IFN-γ secretion in epileptic patients: the influence of co-medication

Carbamazepine is a widely used anticonvulsive agent. Its metabolic pathway leads not only to the major active metabolite, carbamazepine-10,11-epoxide, but also to minor terminal metabolites such as iminostilbene and acridine. Carbamazepine is usually well-tolerated, but it may lead to rare, but serious, hypersensitive reactions associated with hypereosinophilia. The mechanisms of hypersensitivity reactions to carbamazepine are still largely unknown, and the implications of the cell-mediated immune response (Th1 pathway) or the humoral immune response (Th2 pathway) are still not understood in these reactions. It is also unclear whether the parent drug or its subsequent metabolites are the primary trigger agent. In our study, we performed ex vivo experiments to evaluate the stimulation of cytokine secretion by carbamazepine, carbamazepine-10,11-epoxide, iminostilbene and acridine. IL-5, IL-6 and IL-10 were quantified as markers of the Th2 pathway, and IL-2 and IFN-γ were used as markers of the Th1 pathway. Blood samples (n=24) were obtained from epileptic patients routinely treated with carbamazepine alone or co-treated with lamotrigine or valproate. The concentrations of cytokines in the plasma were determined before and after 3h stimulation with drugs. We found a significantly positive effect of co-treatment with valproate on the basal level of IL-5 (p<0.01) and IL-10 (p<0.05). IL-5 production increased only in blood stimulated with a high level of acridine (33µM), whereas IL-6 production was less specifically stimulated (p<0.05). Because IL-5 is the most potent stimulating factor of the eosinophils, we suggest that the potential helper effect of valproate and acridine can lead to hypersensitive reactions to carbamazepine in the context of the humoral immune response.

Hitchhikers on the fungal highway: The helper effect for bacterial migration via fungal hyphae

Previous work in our laboratory showed that several bacterial strains, either singly or in association with other bacteria (community migration), were capable of migrating together with the saprotrophic fungus Lyophyllum sp. strain Karsten through soil microcosms. A possible involvement of the type III secretion system (TTSS) in migration was indicated. In this study, we addressed the basis of the community migration, which might lie in a migration helper effect exerted by particular single-strain migrators on other members of the community. Different culturing (plating) as well as culture-independent (PCR-DGGE) methods were applied to assess the effects of putative bacterial helpers in the migration. We used, as a model, the migration-proficient Burkholderia terrae BS001 as the canonical helper strain. PCR-DGGE analysis of the soil system with or without added strain BS001, revealed that the latter consistently stimulated the migration of different bacterial species through the soil. This was observed both following introduction of the organism to a bacterial community from soil and on the basis of a similar organism that was naturally present. One strain, Dyella japonica BS003, was identified as an avid comigrator with B. terrae BS001, although it appeared to lag behind the latter strain in its migration speed. Further examination of the B. terrae BS001/D. japonica BS003 interaction at Lyophyllum sp. strain Karsten hyphae showed that the presence of the D. japonica strain did not negatively affect the growth and migration of B. terrae BS001 with the fungus. A biofilm of B. terrae BS001 was formed on the fungal hyphal front, and we postulate a role for this biofilm in the migration helper effect.

Breeding ecology of the Chestnut-crowned Babbler: a cooperative breeder in the desert

Cooperative breeding systems provide a rich testing ground for evolutionary theory because of the apparent paradox posed by individuals caring for the offspring of others. Such breeding systems are unusually common among Australian birds, but most species have been studied within the temperate and tropical zones. Here we provide the first detailed study of the breeding ecology of the Chestnut-crowned Babbler (Pomatostomus ruficeps), which was conducted in an arid-zone setting. Chestnut-crowned Babblers bred in groups of 2–13. Breeding occurred between late winter and early summer, 81% of 16 groups fledged offspring and 38% of groups fledged two broods. Clutch-size ranged from two to five, and one to five chicks fledged from successful nests after 21–25 days of provisioning. The size of the breeding unit had a significant effect on breeding success, with an extra chick fledged in the season for every three helpers in the group. These helper effects are among the strongest reported for pomatostomid babblers, perhaps reflecting the extreme environment in which this study was conducted. This study will form a basis for future research into the cooperative breeding system of Chestnut-crowned Babblers and aid with our understanding of variation in the breeding ecology of Australian babblers.

Novel adenovirus-based helper system to support production of recombinant parvovirus

Preclinical studies using various cell culture and animal systems highlight the potential of recombinant rodent parvoviruses (recPVs) for cancer therapy. Production of these viruses is, however, not efficient and this hampers the clinical applications of these agents. In this study, we show that the adenovirus genes E2a, E4(orf6) and VA RNA increase the production of recPVs by more than 10-fold and reduce the time of production from 3 to 2 days in HEK293T cells. The helper effects of these genes can be observed with different recPVs, regardless of the nature and size of the inserted transgene. Furthermore, we generated a recombinant Adenovirus 5 carrying the parvovirus VP transcription unit. This helper, named Ad-VP, allows recPVs to be efficiently produced through a protocol based only on cell infection, making possible to use cell lines, such as NB324K, which are good producers of parvoviruses but are hardly transfectable. Hence, we could further improve viral titers and reduce time and costs of production. This Ad-VP helper-based protocol could be scaled up to a bioreactor format for the generation of the large amounts of recPVs needed for future clinical applications.

Helper effects on pup lifetime fitness in the cooperatively breeding red wolf (Canis rufus).

The evolutionary maintenance of cooperative breeding systems is thought to be a function of relative costs and benefits to breeders, helpers and juveniles. Beneficial effects of helpers on early-life survivorship and performance have been established in several species, but lifetime fitness benefits and/or costs of being helped remain unclear, particularly for long-lived species. We tested for effects of helpers on early- and late-life traits in a population of reintroduced red wolves (Canis rufus), while controlling for ecological variables such as home-range size and population density. We found that the presence of helpers in family groups was positively correlated with pup mass and survival at low population density, but negatively correlated with mass/size at high density, with no relation to survival. Interestingly, mass/size differences persisted into adulthood for both sexes. While the presence of helpers did not advance age at first reproduction for pups of either sex, females appeared to garner long-term fitness benefits from helpers through later age at last reproduction, longer reproductive lifespan and a greater number of lifetime reproductive events, which translated to higher lifetime reproductive success. In contrast, males with helpers exhibited diminished lifetime reproductive performance. Our findings suggest that while helper presence may have beneficial short-term effects in some ecological contexts, it may also incur long-term sex-dependent costs with critical ramifications for lifetime fitness.

The effect of helpers on the postfledging period in a cooperatively breeding bird, the sociable weaver

Understanding the evolution of cooperation requires determining the costs and benefits of cooperative behaviour. In cooperative breeders, where nonbreeding individuals assist in raising offspring, these ‘helpers’ are expected to increase the fitness of breeders and hence empirical research has focused on the effect they have on reproductive output and breeder survival. However, the effects of helpers during the postfledging period are poorly known because of the difficulty of tracking fledglings in the wild. Helper presence might be beneficial for fledglings, for example through continuous food delivery or increased predator vigilance, but potential competition between helpers and fledglings, or changes in investment of parents assisted by helpers, could counteract these positive effects and have a negative influence on postfledging survival probabilities or promote dispersal. We investigated the survival of juvenile sociable weavers, Philetairus socius, raised in pairs alone versus pairs with helpers by using capture–mark–recapture methods to control for individual detectability in survival estimation. We found that local survival in the first year was reduced in young raised by groups versus those raised by pairs. This may reflect either higher mortality or emigration of juveniles raised in groups. Hence, our study reveals significant postfledging effects of cooperative breeding that have not been reported previously and that need to be investigated in studies addressing the evolution of cooperative breeding.

Pseudomonas fluorescens BBc6R8 type III secretion mutants no longer promote ectomycorrhizal symbiosis

The Mycorrhiza Helper Bacterium (MHB) Pseudomonas fluorescens BBc6R8 promotes the ectomycorrhizal symbiosis between Douglas fir roots and Laccaria bicolor. In this study, we identified a non-flagellar type III secretion system (T3SS) in the draft genome of BBc6R8 similar to that described in the biocontrol strain P. fluorescens SBW25. We examined whether this T3SS plays a role in the BBc6R8 mycorrhizal helper effect by creating a deletion in the rscRST genes encoding the central channel of the injectisome. The in vitro effect of BBc6R8 T3SS mutants on the radial growth rate of L. bicolor was unchanged compared with the parental strain. In contrast, T3SS mutants were unable to promote mycorrhization, suggesting that type III secretion plays an important role in the mycorrhizal helper effect of P. fluorescens BBc6R8 independent of the promotion of hyphal growth that BBc6R8 exhibits in vitro.

Group Structure, Nest Size and Reproductive Success in the Cooperatively Breeding Cichlid Julidochromis ornatus: A Correlation Study

AbstractThe effect of group size on reproductive success has long been studied in cooperatively breeding species, as it might provide an adaptive explanation for group‐living in social species. Numerous studies have shown positive effects of subordinates on reproductive success (‘helper effect’), but these studies have also revealed the importance of controlling statistically, or experimentally, for the effect of other factors that might affect reproductive success. Here, we first examine the relationships between group size, body size of group members and nest size in the cooperatively breeding cichlid Julidochromis ornatus, in which unrelated helpers frequently participate in reproduction and their breeding nests inside rock crevices may be crucial for reproduction and survival of all group members. Then, we subsequently investigate the relationship between group size and reproductive success, while controlling for these factors. The results showed that group size was significantly related to body size of group members rather than nest size; and larger breeders had larger helpers. It was found that group size significantly increased group reproductive output. More importantly, reproductive success of male breeders did not depend on the presence of mature helpers, whereas female reproductive success increased when two males assisted her and tended to decrease when two females bred cooperatively. We conclude that breeding groups of J. ornatus have size hierarchical societies that relate to group size, and group composition of genetically unrelated and co‐breeding members affects their reproductive success.

Dendritic cell targeted HIV gag protein vaccine provides help to a DNA vaccine including mobilization of protective CD8 + T cells

To improve the efficacy of T cell-based vaccination, we pursued the principle that CD4(+) T cells provide help for functional CD8(+) T cell immunity. To do so, we administered HIV gag to mice successively as protein and DNA vaccines. To achieve strong CD4(+) T cell immunity, the protein vaccine was targeted selectively to DEC-205, a receptor for antigen presentation on dendritic cells. This targeting helped CD8(+) T cell immunity develop to a subsequent DNA vaccine and improved protection to intranasal challenge with recombinant vaccinia gag virus, including more rapid accumulation of CD8(+) T cells in the lung. The helper effect of dendritic cell-targeted protein vaccine was mimicked by immunization with specific MHC II binding HIV gag peptides but not peptides from a disparate Yersinia pestis microbe. CD4(+) helper cells upon adoptive transfer allowed wild-type, but not CD40(-/-), recipient mice to respond better to the DNA vaccine. The transfer also enabled recipients to more rapidly accumulate gag-specific CD8(+) T cells in the lung following challenge with vaccinia gag virus. Thus, complementary prime boost vaccination, in which prime and boost favor distinct types of T cell immunity, improves plasmid DNA immunization, including mobilization of CD8(+) T cells to sites of infection.

Changes to the Natural Killer Cell Repertoire after Therapeutic Hepatitis B DNA Vaccination

BackgroundImprovements to the outcome of adaptive immune responses could be achieved by inducing specific natural killer (NK) cell subsets which can cooperate with dendritic cells to select efficient T cell responses. We previously reported the induction or reactivation of T cell responses in chronic hepatitis B patients vaccinated with a DNA encoding hepatitis B envelope proteins during a phase I clinical trial.Methodology/Principal FindingsIn this study, we examined changes in the peripheral NK cell populations occurring during this vaccine trial using flow cytometry analysis. Despite a constant number of NK cells in the periphery, a significant increase in the CD56bright population was observed after each vaccination and during the follow up. Among the 13 different NK cell markers studied by flow cytometry analysis, the expression of CD244 and NKG2D increased significantly in the CD56bright NK population. The ex vivo CD107a expression by CD56bright NK cells progressively increased in the vaccinated patients to reach levels that were significantly higher compared to chronically HBV-infected controls. Furthermore, modifications to the percentage of the CD56bright NK cell population were correlated with HBV-specific T cell responses detected by the ELISPOT assay.Conclusions/SignificanceThese changes in the CD56bright population may suggest a NK helper effect on T cell adaptive responses. Activation of the innate and adaptive arms of the immune system by DNA immunization may be of particular importance to the efficacy of therapeutic interventions in a context of chronic infections.Trial RegistrationClinicalTrials.gov NCT00988767

Routes to indirect fitness in cooperatively breeding vertebrates: kin discrimination and limited dispersal

Hamilton demonstrated that the evolution of cooperative behaviour is favoured by high relatedness, which can arise through kin discrimination or limited dispersal (population viscosity). These two processes are likely to operate with limited overlap: kin discrimination is beneficial when variation in relatedness is higher, whereas limited dispersal results in less variable and higher average relatedness, reducing selection for kin discrimination. However, most empirical work on eukaryotes has focused on kin discrimination. To address this bias, we analysed how kin discrimination and limited dispersal interact to shape helping behaviour across cooperatively breeding vertebrates. We show that kin discrimination is greater in species where the: (i) average relatedness in groups is lower and more variable; (ii) effect of helpers on breeders reproductive success is greater; and (iii) probability of helping was measured, rather than the amount of help provided. There was also an interaction between these effects with the correlation between the benefits of helping and kin discrimination being stronger in species with higher variance in relatedness. Overall, our results suggest that kin discrimination provides a route to indirect benefits when relatedness is too variable within groups to favour indiscriminate cooperation.

Wild-Type p53 Enhances Efficiency of Simian Virus 40 Large-T-Antigen-Induced Cellular Transformation

Abortive infection of BALB/c mouse embryo fibroblasts differing in p53 gene status (p53(+/+) versus p53(-/)(-)) with simian virus 40 (SV40) revealed a quantitatively and qualitatively decreased transformation efficiency in p53(-/-) cells compared to p53(+/+) cells, suggesting a supportive effect of wild-type (wt) p53 in the SV40 transformation process. SV40 transformation efficiency also was low in immortalized p53(-/-) BALB/c 10-1 cells but could be restored to approximately the level in immortalized p53(+/+) BALB/c 3T3 cells by reconstituting wt p53, but not mutant p53 (mutp53), expression. Stable expression of large T antigen (LT) in p53(+/+) 3T3 cells resulted in full transformation, while LT expression in p53(-/-) 10-1 cells could not promote growth in suspension or in soft agar to a significant extent. The helper effect of wt p53 is mediated by its cooperation with LT and resides in the p53 N terminus, as an N-terminally truncated p53 (DeltaNp53) could not rescue the p53-null phenotype. The p53 N terminus serves as a scaffold for recruiting transcriptional regulators like p300/CBP and Mdm2 into the LT-p53 complex. Consequently, LT affected global and specific gene expression in p53(+/+) cells significantly more than in p53(-/-) cells. Our data suggest that recruitment of transcriptional regulators into the LT-p53 complex may help to modify cellular gene expression in response to the needs of cellular transformation.

Females increase reproductive investment in response to helper‐mediated improvements in allo‐feeding, nest survival, nestling provisioning and post‐fledging survival in the Karoo scrub‐robinCercotrichas coryphaeus

In many cooperatively‐breeding species, the presence of one or more helpers improves the reproductive performance of the breeding pair receiving help. Helper contributions can take many different forms, including allo‐feeding, offspring provisioning, and offspring guarding or defence. Yet, most studies have focussed on single forms of helper contribution, particularly offspring provisioning, and few have evaluated the relative importance of a broader range of helper contributions to group reproductive performance. We examined helper contributions to multiple components of breeding performance in the Karoo scrub‐robinCercotrichas coryphaeus, a facultative cooperative breeder. We also tested a prediction of increased female investment in reproduction when helpers improve conditions for rearing young. Helpers assisted the breeding male in allo‐feeding the incubating female, increasing allo‐feeding rates. Greater allo‐feeding correlated with greater female nest attentiveness during incubation. Nest predation was substantially lower among pairs breeding with a helper, resulting in a 74% increase in the probability of nest survival. Helper contributions to offspring provisioning increased nestling feeding rates, resulting in a reduced incidence of nestling starvation and increased nestling mass. Nestling mass had a strong, positive effect on post‐fledging survival. Controlling for female age and habitat effects, annual production of fledged young was 130% greater among pairs breeding with a helper, and was influenced most strongly by helper correlates with nest survival, despite important helper effects on offspring provisioning. Females breeding with a helper increased clutch size, supporting the prediction of increased female investment in reproduction in response to helper benefits.

Fungal selectivity of two mycorrhiza helper bacteria on five mycorrhizal fungi associated with Pinus thunbergii

The fungal selectivity of helper effect was revealed using four ectomycorrhizal fungi (Rhizopogon sp., Pisolithus tinctorius, Cenococcum geophilum and Suillus granulatus), and one ectoendomycorrhizal fungus (Wilcoxina mikolae). Previously, we isolated a rhizobacteria, Ralstonia basilensis and Bacillus subtilis, which have the ability to enhance the mycorrhizal symbiosis between S. granulatus and Pinus thunbergii. However, the characteristics of each bacterium on mycorrhizal fungi are still unclear. Therefore, we tried to examine the fungal selectivity of helper effect. A confrontation assay revealed that R. basilensis significantly promoted the in vitro hyphal growth of W. mikolae and S. granulatus, but it had no effect on Rhizopogon sp., P. tinctorius and C. geophilum. These results were consistent with the effects shown by R. basilensis on the mycorrhizal formation of these fungi. On the other hand, B. subtilis promoted the hyphal growth of W. mikolae and C. geophilum but suppressed growth of Rhizopogon sp. B. subtilis significantly stimulated the mycorrhizal formation of S. granulatus. Thus the effects of B. subtilis on hyphal growth and on mycorrhizal formation were inconsistent. These results suggest that R. basilensis and B. subtilis have fungal selective and different mechanisms in their roles as mycorrhiza helper bacteria.

Chaperone-Like Effect of Polyzwitterions on the Interaction of C1q with IgG

The amphiphilic polyzwitterion (PZ) poly(ethylene oxide-b-N,N-dimethyl(methacryloyloxyethyl)ammonium propanesulfonate), zwitterionic surfactant (ZS) n-dodecyl-N,N-dimethyl-3-ammonium-1-propanesulfonate, and zwitterionic monomer (ZM) N,N-dimethyl(methacryloyloxyethyl)ammonium propanesulfonate were analyzed for their suggested chaperone-like effect on the interaction of C1q and IgG. Our results proved that the PZ retarded the C1q interaction with IgG, demonstrating a specific protein-folding helper effect. The ZS enhanced this interaction, when the ZS concentration was lower than the critical micelle concentration (CMC), and retarded it, when the ZS concentration was above the CMC. The ZM, with no self-assembling ability, did not influence this interaction. These results support the hypothesis of a hydrophobic interaction between Pts and hydrophobic domains of partly denatured protein molecules. The amphiphilic self-assemblies, formed by polyzwitterionic macromolecules or zwitterionic surfactants, have the ability to transform the hydrophobic domains of the protein molecules into hydrophilic ones, covering them with their hydrophilic parts.

Helpers and egg investment in the cooperatively breeding acorn woodpecker: testing the concealed helper effects hypothesis

In cooperatively breeding acorn woodpeckers (Melanerpes formicivorus), helper males have a large positive effect on fledging success in good acorn crop years but only a small positive effect in poor acorn crop years, while helper females exhibit the opposite pattern. Based on these findings, we tested the “concealed helper effects” hypothesis, which proposes that laying females reduce investment in eggs (with respect to their size, number, or quality) in a way that confounds helper effects and results in an absence of a relationship between helpers and breeding success. Results generally failed to support the hypothesis. Mean egg size was positively related to temperatures during the 10 days prior to egg-laying and negatively related to the food supply as indexed by the prior fall’s acorn crop, but there were no significant differences vis-à-vis helpers except for interactions with the acorn crop that only partly corresponded to those predicted. With respect to clutch size, females laid larger clutches when assisted by female helpers, opposite the pattern predicted. Although our results suggest that egg size is adjusted to particular ecological circumstances, we conclude that neither egg nor clutch size is adjusted in a way that confounds the apparent effects of helpers, as proposed by the concealed helper effects hypothesis.

Mutual Helper Effect in Copulsing of Dendritic Cells With 2 Antigens

To develop an efficient dendritic cell (DC)-based immunotherapy protocol, we examined whether simultaneous pulsing of DCs with a given antigen and a third-party antigen could enhance their antigen presentation capacity. Purified splenic DCs of Balb/c mice were pulsed separately with immunoglobulin G, ovalbumin, conalbumin, P15 peptide of Mycobacterium tuberculosis, and prostate-specific antigen or double combinations of the aforementioned antigens. In some settings, DCs pulsed with 1 antigen were mixed equally with those pulsed with another antigen. Antigen-pulsed DCs were injected into the footpad of syngeneic mice and proliferation of whole, CD4 and CD8 depleted lymph node cells was measured after restimulation with cognate antigen. Antigen-specific production of interferon-gamma (IFNgamma) was tested in culture supernatants. Frequency of responding lymph node cells was determined by IFNgamma enzyme-linked immunosorbent spot assay. Our results showed that copulsing of DCs with 2 unrelated antigens increased the capacity of DCs to induce antigen-specific T-cell proliferation against both antigens up to 16-fold. Injection of 2 populations of DCs each pulsed with a different antigen, increased proliferation of primed T cells significantly as well. Both CD4 and CD8 depleted populations showed vigorous proliferative response in copulsing system. In addition, copulsing of DCs with 2 antigens resulted in higher frequency of antigen-specific responding cells and significantly more IFNgamma production. Our results clearly showed that unrelated peptides and proteins could be used to enhance efficacy of DC-based vaccines and in this system, each antigen served to help the other one, a condition that we termed as "mutual helper effect."

Intracellular TIAF1 self‐association induces apoptosis

TIAF1 is a small TGF‐beta1‐induced factor, which supports fibroblast growth, T helper cell activation and effect on allograft organ rejection, and probably induces regulator TREG cell differentiation. However, overexpressed TIAF1 induces apoptosis in monocytic and epithelial cells and non‐fibroblasts. Here, we demonstrate that TIAF1 self‐association leads to apoptosis. When cells were overexpressed with CFP‐TIAF1 and YFP‐TIAF1, TIAF1 underwent self‐association and the cells died. Also, TGF‐beta enhances the self‐association for causing apoptosis in TGF‐beta‐sensitive normal epithelial cells. Smad4, an adaptor in the TGF‐beta signaling, binds TIAF1 and blocks the apoptosis. Transiently overexpressed TIAF1 sequesters endogenous Smad proteins in the cytoplasm, and blocks the activation of promoter element driven by Smad proteins. Small interfering RNA‐targeting TIAF1 augments the apoptotic function of overexpressed Smad4. Together, self‐association of TIAF1 induces apoptosis, and that Smad4 interrupts the event.

Enhancement of DNA tumor vaccine efficacy by gene gun–mediated codelivery of threshold amounts of plasmid-encoded helper antigen

AbstractNucleic acid–based vaccines are effective in infectious disease models but have yielded disappointing results in tumor models when tumor-associated self-antigens are used. Incorporation of helper epitopes from foreign antigens into tumor vaccines might enhance the immunogenicity of DNA vaccines without increasing toxicity. However, generation of fusion constructs encoding both tumor and helper antigens may be difficult, and resulting proteins have unpredictable physical and immunologic properties. Furthermore, simultaneous production of equal amounts of highly immunogenic helper and weakly immunogenic tumor antigens in situ could favor development of responses against the helper antigen rather than the antigen of interest. We assessed the ability of 2 helper antigens (β-galactosidase or fragment C of tetanus toxin) encoded by one plasmid to augment responses to a self-antigen (lymphoma-associated T-cell receptor) encoded by a separate plasmid after codelivery into skin by gene gun. This approach allowed adjustment of the relative ratios of helper and tumor antigen plasmids to optimize helper effects. Incorporation of threshold (minimally immunogenic) amounts of helper antigen plasmid into a DNA vaccine regimen dramatically increased T cell–dependent protective immunity initiated by plasmid-encoded tumor-associated T-cell receptor antigen. This simple strategy can easily be incorporated into future vaccine trials in experimental animals and possibly in humans.

220 SYNERGISTIC EFFECT ON EMBRYO DEVELOPMENT BY INCLUSION OF SUPPLEMENTAL EMBRYOS IN AGAR CHIPS

The main scope of this study was to evaluate the likelihood of a helper effect of agar-embedded cleaved embryos on a low number of free embryos at a similar stage of development within the same culture droplet. Such an improved system could be beneficial within ovum pickup/in vitro embryo production (OPU/IVEP) combined protocols whenever a low number of OPU-derived cleaved embryos are produced per donor. Oocytes were recovered from abattoir ovaries, and after in vitro maturation (IVM) and in vitro fertilization (IVF), presumptive zygotes were deprived of cumulus investment and allocated into culture droplets for 24 h. At 48 h from IVF, 4- to 8-cell cleaved embryos were randomly allocated into a control and a treatment group. Control groups consisted of 1, 3, 5, and 10 embryos, respectively, in 50-μL droplets. Treatment groups consisted of 1, 3, and 5 free embryos with the addition of 9, 7, and 5 embryos, respectively, at a similar stage of development embedded in agar chips, so as to reach a total number of 10 cleaved embryos in each culture droplet. Culture was performed for both the control and treatment groups in SOF medium droplets covered with mineral oil, with the supplementation of essential and nonessential amino acids in a controlled gas atmosphere consisting of 5% CO2, 7% O2, and 88% N2 at 39°C. Final embryo output was checked at Day 7 from IVF. When considering only free embryos, the difference in progression to blastocyst development was highly significant between the control and treatment groups: 1) group 1 v. 1 + 9: 6.6 v. 84.3% (P = 0.00000); 2) group 3 v. 3 + 7: 11.1 v. 41.3% (P = 0.00001); 3) group 5 v. 5 + 5: 24.4 v. 42.2% (P = 0.00001). Rate of blastocyst development in the control group containing 10 cleaved embryos was not significantly different from free cleaved embryos in the 3 + 7 (39.2 v. 41.3%, P = 0.71) and 5 + 5 treatment groups (39.2 v. 42.2%, P = 0.54), but was significantly lower when compared with the 1 + 9 treatment group (39.2 v. 84.3%, P = 0.000). For 1, 3, 5, and 10 control group embryos, the numbers of replicates and total cleaved embryos used (n) were 30 (n = 30), 27, (n = 81), 27 (n = 135), and 39 (n = 390), respectively. For the 1 + 9, 3 + 7, and 5 + 5 treatment group embryos, the numbers of replicates and total cleaved embryos used were 32 (n = 32), 29 (n = 87), and 27 (n = 135), respectively. In conclusion, a beneficial effect of agar-embedded embryos on the development of free embryos within the same culture droplet was shown. A striking improvement in late-stage embryo development was particularly evident when considering the 1 v. 1 + 9 control and treatment groups. These results may foster a different strategic approach in in vitro culture to enhance embryo development from highly valuable donors.