Differences In Rate Of Development Research Articles

A test of life‐history theories of immune defence in two ecotypes of the garter snake, Thamnophis elegans

1. Life-history theorists have long observed that fast growth and high reproduction tend to be associated with short life span, suggesting that greater investment in such traits may trade off with self-maintenance. The immune system plays an integral role in self-maintenance and has been proposed as a mediator of life-history trade-offs. 2. Ecoimmunologists have predicted that fast-living organisms should rely more heavily on constitutive innate immunity than slow-living organisms, as constitutive innate defences are thought to be relatively inexpensive to develop and can provide a rapid, general response to pathogens. 3. We present the first study to examine this hypothesis in an ectothermic vertebrate, by testing for differences in three aspects of constitutive innate immunity in replicate populations of two life-history ecotypes of the garter snake Thamnophis elegans, one fast-living and one slow-living. 4. As predicted, free-ranging snakes from the fast-living ecotype had higher levels of all three measures of constitutive innate immunity than the slow-living ecotype. These differences in immunity were not explained by parasite loads measured. Furthermore, both ecotypes exhibited a positive relationship between innate immunity and body size/age, which we discuss in the context of ectotherm physiology and ecotype differences in developmental rates.

An exploration of genome size diversity in dragonflies and damselflies (Insecta: Odonata)

AbstractLike most insect orders, the Odonata (dragonflies and damselflies) remain poorly studied from the perspective of genome size. They exhibit several characteristics that make them desirable targets for analysis in this area, for example a large range in body size, differences in developmental rate, and distinct modes of flight – all of which are related to genome size in at least some animal taxa. The present study provides new genome size estimates and morphometric data for 100 species of odonates, covering about 1/5 of described North American diversity. Significant relationships are reported between genome size and body size (positive in dragonflies, negative in damselflies), and there is also indication that developmental rate and flight are related to genome size in these insects. Genome size is also positively correlated with chromosome number across the order. These findings contribute to an improved understanding of genome size evolution in insects, and raise several interesting questions for future research.

Comparison of the offspring sex ratio between fresh and vitrification-thawed blastocyst transfer

There was a significant difference in the sex ratio between offspring from fresh and from vitrification-thawed blastocyst transfer. The live birth sex ratio influenced by the blastocyst-stage embryo transfer correlated with top-quality embryo morphology.

Effects of the conditioned medium of mesenchymal stem cells on mouse oocyte activation and development

Mesenchymal stem cells (MSCs) have been reported to secrete a variety of cytokines and growth factors acting as trophic suppliers, but little is known regarding the effects of conditioned medium (CM) of MSCs isolated from femurs and tibias of mouse on the artificial activation of mouse oocytes and on the developmental competence of the parthenotes. In the current study, we investigated the effect of CM on the events of mouse oocyte activation, namely oscillations of cytosolic calcium concentration ([Ca(2)+]i), meiosis resumption, pronucleus formation, and parthenogenetic development. The surface markers of MSCs were identified with a fluorescence-activated cell sorter. The dynamic changes of the spindle and formation of pronuclei were examined by laser-scanning confocal microscopy. Exposure of cumulus-oocyte complexes to CM for 40 min was optimal for inducing oocyte parthenogenetic activation and evoking [Ca(2)+]i oscillations similar to those evoked by sperm (95 vs 100%; P > 0.05). Parthenogenetically activated oocytes immediately treated with 7.5 microg/mL cytochalasin B (CB), which inhibited spindle rotation and second polar body extrusion, were mostly diploid (93 vs 6%, P < 0.01) while CB-untreated oocytes were mostly haploid (5 vs 83%, P < 0.01). Consequently, the blastocyst rate was higher in the CB-treated than in the CB-untreated oocytes. There was no significant difference in developmental rate between oocytes activated with CM and 7% ethanol (62 vs 62%, P > 0.05), but the developmental competence of the fertilized oocytes was superior to that of the parthenotes (88 vs 62%, P < 0.05). The present results demonstrate that CM can effectively activate mouse oocytes, as judged by the generation of [Ca(2)+]i oscillations, completion of meiosis and parthenogenetic development.

Tactile stimuli trigger group effects in cockroach aggregations

In many gregarious species, social interactions among group members have such a potent influence on the physiology and/or the behaviour of individuals that isolation has dramatic consequences for their development and survival. Although 'group effects' have been described in many insect species, the mechanisms involved in these processes remain poorly documented. Our aim was to shed light on group effects in cockroaches by investigating the nature of the social stimuli triggering differences in developmental rates between grouped and isolated individuals. Group effects were absent in nonaggregating species (Blattella lituricollis, Blattella biligata), but present in species that form social aggregations (Blattella germanica, Symploce pallens). The presence of conspecifics increased rates of both nymphal development and oothecae production in B. germanica. Tactile stimuli were sufficient to trigger developmental group effects in this gregarious cockroach. Blattella germanica nymphs reared with tactile stimuli, either from a rotating feather or from insects of other species, grew faster than isolated nymphs. Although the role of tactile stimuli has been understudied, they could be involved in group effects in many insect species. Living in groups is widespread and provides different kinds of benefits such as decreased predation risks, decreased energetic costs of movement, increased foraging efficiency or increased encounters with potential mates (Krause & Ruxton 2002). Although a large variety of animals aggregate, the mechanisms responsible for group formation and complexity of communication differ between species (Parrish & Hamner 1997). Depending on these characteristics, conspecifics can have such a potent influence on the physiology and/or behaviour of individuals that social isolation has dramatic consequences for their development and survival (e.g. Harlow 1965; Wilson 1971; House 2001; Zeigler & Marler 2004; Perello a et al. 2006). Two main categories of aggregations emerge from the many definitions that have been given (Parrish et al. 1997). (1) In nonsocial aggregations, individuals are attracted by a given source but do not interact socially and they disperse when the source is consumed or vanishes. In this case, large increases in density at a source can induce microenvironmental modifications that happen to favour development. For example, under crowded conditions some dipteran and lepidopteran nymphs benefit from better regulation of body temperature and of water evaporation than singletons and thus their developmental rates increase (Clark & Faeth 1997; Slone & Gruner 2007). These differences between grouped and isolated individuals have been termed 'mass effects' and are generally observed in large groups of animals (Grasse 1946). (2) In contrast, social aggregations are formed and maintained by mutual attraction among their socially interacting members. In this case, differences in physiological , morphological or behavioural traits between grouped and isolated individuals are mainly attributable to social interactions and have been called 'group effects' (Grasse 1946). They differ from mass effects in that they are caused by the perception of social stimuli emanating from conspecifics and can be observed as soon as only two individuals are grouped (Grasse 1946). Group effects have been described in many insects including orthopterans, aphids, coleopterans and blattodeans; their best-documented consequences pertain to modifications of developmental rates or group-induced phenotypic plasticity. Grouping often accelerates nymphal development (Chauvin 1946; Long 1953), although it can delay imaginal moulting in a few

Sex-specific costs of hatching last: an experimental study on herring gulls (Larus argentatus)

An organism’s pattern of development can have important long-term fitness effects. In species where the sexes differ in size or other phenotypic traits, they may also have different optimal developmental rates. This influences both parental sex allocation strategies and susceptibility of the sexes to early developmental conditions. However, sex differences in developmental rate and vulnerability to environment during the embryonic period are not well understood. In birds, sibling competition and hatching asynchrony may select for accelerated embryonic development of the last offspring in order to reduce their competitive disadvantage after hatching. They may advance their hatching in response to vocal stimuli by the older siblings. It is, however, unclear whether this flexibility in developmental rates is sex specific. In this study, we experimentally manipulated between-embryo contact and tested whether this affected the pre-natal developmental rate and post-hatching performance of male and female offspring from last-laid eggs in the herring gull. Post-hatching performance was measured both in competitive and non-competitive situations. Among young incubated in isolation, males hatched faster than females, but both sexes fledged in similar, relatively good condition. Among young incubated with normal between-embryo contact, hatching time did not differ between sexes, but males fledged in poorer condition than females, regardless of whether they were reared singly or in a brood. These results suggest that male and female offspring differ in their ability to mitigate the costs of hatching asynchrony.

Anueploidy not gender impacts the developmental rate of IVF-derived human embryos as confirmed by day 3 blastomere biopsy and preimplantation genetic diagnosis

Anueploidy not gender impacts the developmental rate of IVF-derived human embryos as confirmed by day 3 blastomere biopsy and preimplantation genetic diagnosis

Intermittent hypoxia in eggs ofAmbystoma maculatum: embryonic development and egg capsule conductance

The spotted salamander Ambystoma maculatum breeds in shallow freshwater pools and imbeds its eggs within a common outer jelly matrix that can limit oxygen availability. The eggs are impregnated with the unicellular alga Oophilia amblystomatis, which produces oxygen during the day but consumes oxygen at night. This daily cycle of algal oxygen production drives a diurnal fluctuation of oxygen partial pressure (PO2) within the eggs, the magnitude of which depends on the distance of an egg from the exterior of the jelly matrix and on the ambient PO2 of the pond. We subjected A. maculatum eggs to fluctuating oxygen levels with a variable minimum PO2 and an invariable maximum, to simulate natural conditions, and measured differences in developmental rate, day and stage at hatching, and egg capsule conductance (GO2). Lower minimum PO2 slowed development and resulted in delayed, yet developmentally premature hatching. GO2 increased in all treatments throughout development, but PO2 had no detectable effect on the increase. Intermittent hypoxia caused comparable but less pronounced developmental delays than chronic hypoxia and failed to elicit the measurable change in GO2 seen in ambystomatid salamander eggs exposed to chronic hypoxia.

Differential responses of honeybee (Apis mellifera) patrilines to changes in stimuli for the generalist tasks of nursing and foraging

Which task a social insect worker engages in is influenced by the worker’s age, genotype and the colony’s needs. In the honeybee, Apis mellifera, genotype influences both the age a worker switches tasks and its propensity of engaging in specialist tasks, such as water collecting, which only some workers will perform. In this study, we used colonies with natural levels of genetic diversity and manipulated colony age demography to drastically increase the stimuli for the generalist tasks of foraging and nursing, which all workers are thought to engage in at some point in their lives. We examined the representation of worker patrilines engaged in nursing and foraging before and after the perturbation. The representation of patrilines among foragers and nurses differed from that of their overall colony’s population. In the case of foraging, over- and underrepresentation of some patrilines was not simply due to differences in rates of development among patrilines. We show that replacement foragers tend to be drawn from patrilines that were overrepresented among foragers before the perturbation, suggesting that there is a genetic component to the tendency to engage in foraging. In contrast, the representation of patrilines in replacement nurses differed from that in the unperturbed nursing population. Our results show that there is a genetic influence on even the generalist tasks of foraging and nursing, and that the way patrilines in genetically diverse colonies respond to increases in task stimuli depends upon the task. The possible significance of this genetic influence on task allocation is discussed.

Dynamic Changes in Microtubules and Early Development of Reconstructed Embryos after Somatic Cell Nuclear Transfer in a Non-Human Primate

In order to improve somatic cell nuclear transfer (SCNT) efficiency and to understand cellular changes in SCNT, the dynamic changes in microtubules/DNA and early development of SCNT embryos with single or multiple pronuclei were investigated, along with activation timing on efficiency of SCNT, were studied in the Cynomolgus monkey. The confocal images showed that microtubules assembled around condensed DNA at 1h after cell injection; normal or abnormal reconstructed spindle formed at 2 h after cell injection; and reconstructed spindle separated at 2 h after activation. The results of nuclear formation showed that 61.3% of the reconstructed embryos did not form pronuclei; 19.3% formed a single nucleus, and 11.9% and 7.5% formed two and more than two reconstructed pronuclei, respectively. The cleavage and 8-cell development rates of SCNT embryos with pronuclei were significantly higher than those without pronuclei, but there was no difference in development rates among NT embryos with single, two and more then two pronuclei. Activation at 2 h after cell injection yielded more embryos with pronuclei and yielded 8-cell NT embryos more reliably than did activation at 3-4 h. In conclusion, microtubules assembled around condensed DNA at 1-2 h after cell injection, and formed a spindle at 2 h after SCNT, which separated at 2 h after activation; early development was affected by activation time, but no different between single and multiple pronuclei.

The relevance of life-history traits in the establishment of the invader Eudiaptomus gracilis and the extinction of Eudiaptomus padanus in Lake Candia (Northern Italy): evidence for competitive exclusion?

The niches invaded by exotic species are generally not entirely vacant, and one possible factor affecting the success of the invader to establish a permanent large population may be the ability of the former to outcompete native species. Eudiaptomus gracilis, which was not present in Italy before the 1980s, is becoming established in an increasing number of Northern Italian lakes, ultimately replacing the endemic E. padanus. Coexistence of the two species in Lake Candia lasted only 7 months, suggesting that species replacement was determined either by environmental changes or by strong competition. To assess whether the potential for interspecific competition existed and to identify species' traits which could explain the competitive superiority of E. gracilis, we examined field seasonal patterns, reproductive parameters and body size of the two species. E. padanus abundance was probably reduced by predation and parasitism, which favoured the displacement of the species shortly after invasion by E. gracilis. The temporarily underexploited niche provided an opportunity for the successful establishment of the invader. The reproductive patterns of the two species were found to be similar in most of their features, with the exception of a markedly larger clutch size and a smaller egg volume in the resident species. By contrast, the invader showed a higher adult:egg ratio and a lower death rate. Thus, despite the greater fecundity of E. padanus, the competitive success of the invader might be attributable to interspecific differences in developmental rates and/or juvenile mortality.

Use of androgenesis for estimating maternal and mitochondrial genome effects on development and oxygen consumption in rainbow trout, Oncorhynchus mykiss

Chromosome set manipulation was used to produce rainbow trout, Oncorhynchus mykiss, with identical nuclear backgrounds, but different maternal backgrounds to determine mitochondrial effects on development rate and oxygen consumption. Significant differences in development rate and oxygen consumption were observed between groups from different females. Development rates ranged from a mean of 317.97 degree days (°d) to 335.25 °d in progeny from different females. Mean oxygen consumption rates ranged from 3.31 μmol O 2 g − 1 wet mass h − 1 to 9.66 μmol O 2 g − 1 wet mass h − 1 . Oxygen consumption and development rate analysis revealed the two slowest developing groups had the highest oxygen consumption rates. Development rate differences between second generation clonal females indicate that mitochondrial genomes play a significant role on early development and are comparable to development rate differences between clonal lines of rainbow trout. These results indicate that selection for mitochondrial genomes could increase growth rates and possibly food conversion ratios in aquaculture species.

On the power of multivariate latent growth curve models to detect correlated change.

We evaluated the statistical power of single-indicator latent growth curve models (LGCMs) to detect correlated change between two variables (covariance of slopes) as a function of sample size, number of longitudinal measurement occasions, and reliability (measurement error variance). Power approximations following the method of Satorra and Saris (1985) were used to evaluate the power to detect slope covariances. Even with large samples (N = 500) and several longitudinal occasions (4 or 5), statistical power to detect covariance of slopes was moderate to low unless growth curve reliability at study onset was above .90. Studies using LGCMs may fail to detect slope correlations because of low power rather than a lack of relationship of change between variables. The present findings allow researchers to make more informed design decisions when planning a longitudinal study and aid in interpreting LGCM results regarding correlated interindividual differences in rates of development.

Effect of Exposure Duration of Ovaries and Oocytes at Ambient Temperature on Parthenogenetic Development of Porcine Follicular Oocytes

This study was conducted to evaluate the effects of exposing porcine ovaries to 30-33 C during transportation for 4 h and subsequently room temperature (25 C) for 6 h of storage on in vitro maturation (IVM) and subsequent parthenogenetic development of oocytes collected from the ovaries. After IVM, oocytes having a tight oopalsm membrane and no signs of degeneration were exposed to Dulbecco's phosphate-buffered saline (DPBS) with 7% ethanol (v/v) for 7 min to induce parthenogenetic activation. Moreover, we also determined whether exposure of the collected oocytes to room temperature for 1, 2 and 4 h in DPBS or porcine follicular fluid (pFF) affected parthenogenetic development. When porcine ovaries were stored after transportation, oocytes collected from the stored ovaries showed a significantly higher rate of degeneration after 65 h of IVM (58.4%) and a significantly lower rate of cleavage after parthenogenetic activation (40.1%) than oocytes collected from ovaries immediately after transportation (38.9% and 47.4%, respectively). However, there was no significant difference in developmental rates to the morula and blastocyst stages between these two groups (14.4% and 14.3%, respectively). The duration of preservation, 1, 2, and 4 h, of oocytes in DPBS did not affect parthenogenetic development. In contrast, when preserved for 4 h in pFF, the developmental rates of the oocytes were significantly decreased. This suggested that some factor(s) in follicular fluid affects the developmental rate of oocytes with the passage of time in ambient conditions. These results suggest that even after 6 h storage of ovaries, oocytes having normal morphology after IVM have the same rate of parthenogenetic development as oocytes collected from ovaries just after 4 h of transportation, except for a lower cleavage rate, and that exposure of oocytes to room temperature for 4 h in DPBS does not affect their parthenogenetic developmental competence.

Relationship between hypothalamic-pituitary-adrenal axis responsiveness and age, sexual maturity status, and sex in Japanese quail selected for long or short duration of tonic immobility

The influences of age (4 to 12 wk), sexual maturity status, and sex on hypothalamic-pituitary-adrenal (HPA) axis responsiveness were investigated by measuring changes in peripheral basal levels of corticosterone (B) and responses to 10 min of physical restraint in a crush cage or injection of 1-24 adrenocorticotropic hormone (ACTH) in 2 genotypes of Japanese quail divergently selected for long (LTI) or short (STI) duration of tonic immobility (TI). Although gonad development was more advanced in male STI quail, most birds were still immature at 4 wk of age, but sexual maturity was fully acquired by 6 wk of age in both sexes and genotypes. This change was associated with increases in basal B levels in both genotypes and sexes. On the other hand, HPA axis responsiveness to restraint and adrenal responsiveness to 1-24 ACTH injection remained stable in STI quail, whatever the age. Conversely, significant responses to restraint compared with basal B levels were only observed at 4 and 6 wk of age, and adrenal responsiveness increased with age in LTI quail. Moreover, higher B levels were measured in response to restraint in STI than in LTI quail, whereas similar adrenal responses were measured at 9 and 12 wk of age. We concluded that an increase in basal B levels is associated with the stage of sexual maturity acquired, but it did not affect HPA axis responsiveness or adrenal B response capacity. On the other hand, age affected HPA axis responsiveness and adrenal B response capacity in LTI quail of both sexes but not in STI quail. It is hypothesized that functional HPA axis maturity occurs after 6 wk of age in the LTI genotype, but it is reached by 4 wk of age in the STI genotype. In conclusion, the divergent selection program for TI conducted on quail resulted in changes in HPA responsiveness that probably resulted from differences in development rate and function of the adrenal glands or other upstream structures of the HPA axis.

Hahb-10, a Sunflower Homeobox-Leucine Zipper Gene, is Regulated by Light Quality and Quantity, and Promotes Early Flowering when Expressed in Arabidopsis

Homeodomain-leucine zipper proteins constitute a family of transcription factors found only in plants. Expression patterns of the sunflower homeobox-leucine zipper gene Hahb-10 (Helianthus annuus homeobox-10), that belongs to the HD-Zip II subfamily, were analysed. Northern blots showed that Hahb-10 is expressed primarily in mature leaves, although expression is clearly detectable in younger leaves and also in stems. Considerably higher expression levels were detected in etiolated seedlings compared with light-grown seedlings. Induction of Hahb-10 expression was observed when seedlings were subjected to treatment with gibberellins. Transgenic Arabidopsis thaliana plants that express Hahb-10 under the 35S cauliflower mosaic virus promoter show special phenotypic characteristics such as darker cotyledons and planar leaves. A reduction in the life cycle of about 25% allowing earlier seed collection was also observed, and this phenomenon is clearly related to a shortened flowering time. When the number of plants per pot increased, the difference in developmental rate between transgenic and non-transformed individuals became larger. After gibberellin treatment, the relative difference in life cycle duration was considerably reduced. Several light-regulated genes have been tested as possible target genes of Hahb-10. One of them, PsbS, shows a different response to illumination conditions in transgenic plants compared with the response in wild-type plants while the other genes behave similarly in both genotypes. We propose that Hahb-10 functions in a signalling cascade(s) that control(s) plant responses to light quality and quantity, and may also be involved in gibberellin transduction pathways.

Clone of Chinese Jinan redcross yellow cattle and evaluation of reproductive characteristics of cloned calf

Somatic cell clone technology is a viable approach to preserving endangered livestock and wildlife genetic resources. In the present research, somatic cell nuclear transfer (SCNT) was performed using granulose cells from the critical endangered Chinese red-cross yellow cattle as donor cells. A total of 211 oocytes were manipulated and 166 (79%) of them were successfully enucleated. 112 (67.4%) SCNT embryos were reconstructed, 94 (83%) of them cleaved, and 48 (43%) of them developed to blastocyst stage. SCNT blastocysts were transferred to 6 Holstein recipients, and 2 (33%) of them were found to be pregnant. One of them maintained to term and delivered a calf, whereas another aborted. Effect of different fusion buffer (mannitol vs. Zimmerman fusion buffer) and different activation methods (calcium ionophore+6-DMAP vs. cycloheximide+CB) on fusion rate and development of SCNT embryos were investigated. The results indicated that: (i) on condition of two DC pulses of 2.5 kV/cm for 10 μs each, fusion rates were higher in mannitol solution than in Zimmerman fusion buffer (71% vs. 61%, respectively, p 0.05); (ii) There was no significant difference in development rates to the blastocyst stage for SCNT embryos activated by calcium ionophore+6-DMAP or by cycloheximide+CB (42% vs. 46%, respectively, p>0.05). Microsatellite DNA analysis examining 28 loci confirmed that the cloned calf was genetically identical to the donor Jinan red-cross yellow cattle and different from the recipient females. Growth and reproductive performance of cloned cow were evaluated, and there were no differencei cross-red n it between cloned and normal control Jinan yellow cattle. Furthermore, the cloned yellow cow has delivered a healthy yellow calf.

Cost and mitigation of insecticide resistance in the maize weevil, Sitophilus zeamais

Abstract. A common assumption in models of insecticide resistance evolution is the association between resistance and fitness costs in the absence of insecticides. There is empirical evidence of such associations, but their physiological basis (and mitigation) is little investigated. Pyrethroid‐resistant populations of the maize weevil Sitophilus zeamais (Coleoptera: Curculionidae) offer this opportunity. Pyrethroid resistance in this species was initially observed in five Brazilian states by 1995, but the phenomenon apparently decreased and did not spread to other regions, probably due to the occurrence of a fitness disadvantage in resistant individuals in the absence of insecticides. The present investigation aims to verify whether differences in respiration rate and fat body morphology are related to differences in rate of development in Brazilian populations of S. zeamais resistant to insecticides, and thereby provide evidence for the existence (or not) of a physiological fitness cost acting against insecticide resistance in maize weevils. This may occur due to a possible energy trade‐off between insecticide resistance and other physiological processes associated with development and reproduction. To achieve this, studies of the rate of development, respiration and fat body cytomorphology are carried out in one insecticide‐susceptible (from Sete Lagoas) and two resistant populations (from Jacarezinho and Juiz de Fora) of S. zeamais. The resistant population from Jacarezinho shows that higher body mass is associated with higher energy reserves (larger trophocyte area) for development and reproduction, as well as for insecticide resistance. However, the resistant population from Juiz de Fora does not appear to have large enough energy allocation for insecticide‐resistance expression and development and/or reproductive performance, suggesting a trade‐off between resistance and other life history traits.

320 USE OF SYNTHETIC HYALURONAN OR POLYVINYLPYRROLIDONE FOR INTRACYTOPLASMIC SPERM INJECTION INTO MOUSE OOCYTES

The use of polyvinylpyrrolidane (PVP) in intracytoplasmic sperm injection (ICSI) seems to be exclusively related to its surfactant and colloidal properties. In contrast to PVP, which can be toxic to mouse embryos, hyaluronan (HA) is a biological compound. In addition to its colloidal property, HA plays an important biochemical role in cell proliferation and migration and can be found intracellularly in the cleaving stage of mouse, sheep and primate embryos (Hunter RHF 1994 Mol. Reprod. Dev. 39, 176–181). We expect that the viscoelastic properties of HA in combination with its physiological functions may benefit the ICSI procedure. Oocytes at MII stage were collected from CD-1 mice 14 h after hCG injection (h-pi) and were kept at 37°C in KSOM medium for 30 min before ICSI. Semen used for injection was frozen by direct plunge into liquid nitrogen in M2 medium without cryoprotectants. Samples were thawed at 25°C in the air and mixed (1:5) with M2 medium containing either 10% PVP; 360000 MW (w/v; Sigma, St. Louis, MO, USA) or 60% (v/v) synthetic HA (s-HA; MAP-5; Bioniche Inc, Belleville, ON Canada) with comparable viscosity. Injections were performed at 25°C using a mercury-containing pipette attached to a piezo impact unit (Prime Tech, Ibaraki, Japan). A total of 239 oocytes (115 PVP and 124 s-HA) were injected in groups of ten in four replicates. Individual sperm heads decapitated by the freeze/thaw procedure were injected into oocytes and kept for 15 min at 25°C. Oocytes that survived ICSI were placed in 35 μL drops of KSOM medium (∼15 zygotes per drop) under paraffin oil at 37°C and 5% CO2 in humidified air. Cleavage and developmental rates were recorded at 24, 48, and 96 h after oocyte injection. Embryos which developed to the blastocyst stage were transferred to pseudo-pregnant females mated with vasectomized males. At Day 13, recipient mice were sacrificed and the number of implantations and fetuses were recorded. Data were compared between groups by Chi-square analysis. Significantly (P &lt; 0.05) more embryos survived ICSI in PVP (74%) than in s-HA group (56%), which was primarily related to sperm adhesiveness to the injection pipette. However, there were no differences in developmental rates at any stage of in vitro embryo culture between groups (2 cell, 93 vs. 100%; 4–8 cell, 100 vs. 100%; blastocyst, 44 vs 50%) for PVP and s-HA, respectively. Significant differences (P &lt; 0.05) between groups were observed in embryo implantation rates. When ICSI was performed with s-HA, 29 out of 35 blastocysts (83%) transferred to synchronized recipients were implanted, which was accomplished only by 19 of the 35 from the PVP group (54%). However, there was no difference between groups in the number of fetuses detected (8 (23%) vs. 9 (26%) for PVP and s-HA, respectively). The use of s-HA for mouse ICSI can be a valuable alternative to PVP. Hyaluronan may show further benefit if sperm adhesiveness to the micropipette can be eliminated, and may be superior to PVP if embryo implantation rates in the s-HA group can be sustained. The authors would like to thank Bioniche, Inc., Belleville, ON, Canada for donating MAP-5.

248 EMBRYO DEVELOPMENT IN MICRODROPS AND MICROCHANNELS: COMPARISON OF NCSU23 SEQUENTIAL AND NON-SEQUENTIAL CULTURE MEDIUM

The in vitro culture systems used to produce pig embryos generally result in few embryos developing to the blastocyst stage. The use of pyruvate (pyr) and lactate (lac) during the culture of zygotes to the 8-cell stage followed by glucose (glu) supplementation replacing pyr and lac appears to be beneficial for embryo development in the pig. The aim of this study was to compare the embryo development rates from pig oocytes fertilized with and without cumulus cells in 100-μL microdrops (MD) and cultured in 100-μL MD or microchannels (MC), using NCSU23 containing 8 mg/mL of BSA and supplemented with (1) glu or (2) pyr/lac or (3) pyr/lac for the first three days and then with just glu for the remainder of culture period (pyr/lac-glu). Sow oocytes were matured in TCM199 supplemented with gonadotropins for the first 22 h, and for an additional 22 h without hormones. After 44 h of maturation, oocytes were placed in MD of modified tris-buffered medium to be fertilized using 3 × 105 sperm/mL. Oocytes were divided into two groups for fertilization: with and without cumulus cells. Following 6 h of fertilization, all inseminated oocytes were washed, divided into groups of 15, allotted to the three culture media treatment groups as described above, and incubated in either MD or MC. With the exception of one treatment there were no significant differences in development rates among embryos cultured in MD or MC, hence data were pooled from these two culture devices. Only oocytes fertilized without cumulus cells and cultured in pyr/lac in MC appeared to have lower rates of blastocyst formation (11.67%) than those cultured in MD (26.67%) in the same culture medium. When the six treatments were compared, oocytes fertilized with cumulus cells and cultured in glu had significantly higher (P &lt; 0.05) blastocyst rates and hatching rates compared with the other treatments, with the exception of those fertilized without cumulus cells and cultured in pyr/lac-glu. There were no significant differences among other treatments in Day 7 blastocyst or in Day 9 hatching rates. In conclusion, both culture devices can be used to reach similar blastocyst rates with different treatments. In this experiment, the removal of cumulus cells before fertilization appeared to enhance embryo development in vitro when sequential media are used. On the other hand, the presence of cumulus cells before fertilization seems to enhance embryo development when non-sequential glu medium is used. Table 1. Embryo development rates on Day 9 for three different culture treatments