Higher Number Of Embryos Research Articles

국내 육성 품종 '탐미유채'의 소포자 배양 시 배양조건이 배발생에 미치는 영향

국내 육성 유채 품종의 소포자 배양을 통한 효율적인 소포자배 생산법의 확립을 위해 '탐미유채'를 대상으로 소포자 배양 시 적당한 꽃봉오리의 크기, 고온처리시간, 배양밀도, 기내 증식조건 등의 배양조건을 구명하고자 실시한 결과, 소포자배 발생은 1핵기말과 2핵기 초기 상태의 소포자가 포함된 3.0~3.5 mm 크기의 꽃봉오리에서 채취한 소포자에서 배발생률이 가장 높았으며, 배지 1 mL 당 약 388개의 소포자 배가 발생하였다. 배양 초기에 <TEX>$32.5^{\circ}C$</TEX>에서 2일간 열처리하였을 때 배발생률이 가장 높았다. 소포자의 배양밀도에 따른 배발생은 <TEX>$5{\times}10^4$</TEX>개/mL일 때 가장 높았으며, 배양밀도가 더 이상 높아지면 배발생을 억제하였다. 발생한 소포자배는 <TEX>$0.5mg{\cdot}L^{-1}$</TEX> NAA와 <TEX>$1mg{\cdot}L^{-1}$</TEX> BA가 첨가된 MS 고체배지에 치상하여 배양하면 정상적인 신초로 재분화되었고, 발달한 신초를 절취하여 식물성장 조절제가 첨가되지 않은 MS고체배지에서 배양하면 뿌리가 발달하여 정상적인 식물체로 성장하였다. For the establishment of an efficient embryogenesis from microspore culture in Brassica napus L. domestic cultivar 'Tammiyuchae', four different factors affecting microspore embryogenesis and plantlet regeneration were investigated. The highest embryogenesis rate was achieved when microspores at late uninucleate to early binucleate stage were isolated from flower buds with a length of 3.0~3.5 mm. On average, 388 embryos generated from 1 ml of microspores media. The highest number of embryos was obtained when microspores were subjected to <TEX>$32.5^{\circ}C$</TEX> for 2 days. Embryogenesis of 'Tammiyuchae' was increased with increasing microspore culture density up to about <TEX>$5{\times}10^4ea/mL$</TEX>. Gradually higher culture density repressed embryogenesis of microspores. Regeneration rate of shoots from microspore-derived embryos was observed in MS solid medium supplemented with <TEX>$0.5mg{\cdot}L^{-1}$</TEX> NAA and <TEX>$1.0mg{\cdot}L^{-1}$</TEX> BA, and grew well in MS solid medium without plant growth regulators.

314 RELATIONSHIP BETWEEN SERUM ANTI-MULLERIAN HORMONE (AMH), OVARIAN RESERVE, AND EMBRYO PRODUCTION IN SUPEROVULATED HOLSTEIN COWS

The relations between serum anti-Mullerian hormone (AMH), oocyte numbers, and in vivo embryo production in Holstein heifers were evaluated. The AMH levels of 15 unstimulated cows were followed at weekly intervals during their oestrous cycles and monthly for 4 months. Forty-one superovulated heifers were evaluated at ovum pick-up (OPU) performed 20 h after cystorelin administration, and 125 others were evaluated at embryo recovery. Animals were followed over 3 consecutive cycles induced using a modified Ovsynch protocol with 4 days of FSH (Pluset H, Minitube of America, Verona, WI, USA). Blood samples were collected in serum tubes and spun within 2 h. The samples were stored at –20°C until evaluation using the Minitube of America AMH-bovine specific immunoassay (AMH Fertility Assay™). The statistical analyses (ANOVA and data correlation) were performed using Statview 5 with P &lt; 0.05. Serum AMH ranged from 43 to 960 pg mL–1. The average AMH level of all cows was stable during the oestrous cycle and for each of the 4 monthly measurements. There was a high correlation between all values per animal (r2 = 0.9077; P &lt; 0.01), suggesting that AMH levels are consistent during the cycle and for at least 4 consecutive months. Animals that were repeatedly stimulated showed decreasing AMH levels (509 ± 295, 299 ± 210, 211 ± 119) and a decrease in recovered embryos (7.4 ± 4, 5.6 ± 3.8, 4.2 ± 3.2; P = 0.02). The number of oocytes was not altered by multiple stimulations (10.4 ± 9.8, 11.3 ± 6.2, 8.5 ± 7.6; P = 0.75). As AMH and embryo numbers decreased after multiple stimulations, only the first AMH value and results of the first OPU or flush were used to establish following correlation. Serum AMH showed a positive correlation to the number of oocytes (r2 = 0.245) and embryos collected (r2 = 0.27).When separated into AMH categories, low (&lt;100), normal (100–400), and high (&gt;400 pg mL–1), high-AMH OPU animals yielded significantly higher numbers of oocytes than the animals in the normal or low AMH groups (13.8 ± 9.2 v. 9.2 ± 5.2 and 5.6 ± 3.9; P = 0.001). Flushed animals with high AMH levels had significantly higher numbers of embryos than those with low AMH (10.9 ± 7.9 v. 5.7 ± 5; P = 0.002). Comparison of the first AMH value to the average number of oocytes or embryos collected over the course of 3 collections/animal showed a positive correlation to the average number of oocytes/collection from individual OPU donors (r2 = 0.436) and a positive correlation to the average number of embryos/collection from individual donors (r2 = 0.176). When separated into AMH groups, high-AMH flushed animals had significantly higher numbers of embryos than the normal- or low-AMH animals (9.3 ± 3.1 v. 5.7 ± 3.4 and 4.5 ± 2; P = 0.0001). As OPU animals with low AMH were used only once, average oocyte/collection data was not available for this category. A significant difference was observed between the high- and normal-AMH categories (12 ± 3.6 v. 7 ± 2; P = 0.0001). Circulating AMH is stable over time in unstimulated animals but decreases in repetitively stimulated animals. Anti-Mullerian hormone is highly associated with superovulation response and oocyte and embryo production, and its use should improve animal selection to achieve improve efficiency of multiple-ovulation embryo transfer.

DETERMINATION OF THE OPTIMUM MICROSPORE DEVELOPMENT STAGE AND OPTIMUM CULTURE MEDIUM IN ASPARAGUS (ASPARAGUS OFFICINALIS VAR. ALTILIS L.) FOR ANTHER CULTURE

Asparagus is a dioecious vegetable. The main target of asparagus breeders is to obtain hybrids composed of exclusively male plants, since they are more productive than female ones. Anther culture is a method to obtain haploids that can be doubled with colchicine treatment. In order to determine the optimal microspore development stage for androgenesis, anthers were stained with ethidium bromide and examined under fluorescence microscope. Anthers with microspores at the late uninucleate stage were cultured on Murashige and Skoog (MS) basal medium supplemented with three growth regulators at different concentrations (1 mg L-1 2.4 D + 0.1 mg L-1 BA + 0.5 mg L-1 NAA; 1 mg L-1 BA + 5 mg L-1 NAA; 0.5 mg L-1 BA + 1 mg L-1 NAA). The highest number of embryos (8.33%) and the number of plantlets (13.33%) were obtained on MS medium with 0.5 mg L-1 BA and 1 mg L-1 NAA.

Efecto de la edad y la hora de la aspiración sobre la calidad oocitaria y el desarrollo in vitro en hembras bovinas prepúberes

Objetivo. Este estudio evaluó el efecto de la edad y el período de aspiración folicular so­bre la calidad del oocito y su posterior desarrollo in vitro con donadoras prepúberes Bos indicus mestizas. Materiales y métodos. Las hembras donantes recibieron un implante de norgestomet y benzoato de estradiol y cuatro días más tarde una dosis única de 150 UI de eCG. Las aspiración folicular se realizó en tres tiempos (24, 48, y h 56) y en cuatro edades (3, 6,10, y 12 meses). Todos los folículos antrales fueron aspirados y se realizaron procedimientos de maduración, fertilización y cultivo in vitro. Resultados. Se aspiraron 439 folículos en total, de los cuales 385 (87.7%) fueron de 2-5 mm, 41 (9.33%) fueron de 5-10 mm, y fueron 13 (2.3%) &gt; 10 mm. Se recuperó un total de 373 oocitos (84.9%) y se encontraron diferencias significativas (p&lt;0.05) entre los promedios de los animales de 6 (9.3) y 10 meses (32.3). 285 (76.4%) oocitos fueron sometidos a los procesos in vitro. La tasa de división fue significativamente mayor (p&lt;0.05) en los animales de 10 (27.1%) y 12 meses (26.8%). Aunque el número de embriones transferibles fue bajo, existieron di­ferencias entre las edades (p&lt;0.05), obteniendo el porcentaje más alto los animales de 10 meses (12.6%). Conclusiones. Un período de aspiración superior a 24 h tiene un efecto negativo sobre la calidad del oocito. Algunos oocitos procedentes de terneras de 3 meses fueron competentes en el desarrollo in vitro, sin embargo, un mayor número de embriones se produjeron con hembras de 10 y 12 meses de edad.

Involvement of ethylene in somatic embryogenesis in Scots pine (Pinus sylvestris L.)

The involvement of the plant growth regulator ethylene in somatic embryo maturation of Pinus sylvestris (L.) was investigated. Genes that encoded 1-aminocyclopropane-1-carboxylate synthase (ACS), the rate-limiting enzyme in the ethylene biosynthesis pathway, were isolated and characterized. Two novel complementary DNAs (cDNAs) of PsACS1 and PsACS2 that encode ACS were isolated from embryogenic cultures (ECs) along with their polymerase chain reaction (PCR) products. The two sequences shared 69% similarity at amino acid level. PsACS1 was expressed in ECs at the proliferation and maturation stages, whereas the PsACS2 transcript was expressed in embryos only at the maturation stage. These expression levels varied among the five P. sylvestris genotypes studied. Moreover, the high levels of PsACS2 transcript abundance correlated with higher numbers of embryos at stage 3 of development. Ethylene production reached the highest levels at stage 3, and subsequently dropped. Hence, expression of PsACS2 corresponded to ethylene production during embryo development and could thus serve as a genetic marker for the early maturation stage. Both gene expression and ethylene production varied among the different genotypes. Investigating expression of ACC synthase genes further enhanced our understanding of the role of ethylene in embryo development and maturation and improved the efficiency of somatic embryogenesis of P. sylvestris.

High rate of biological loss in assisted reproduction: it is in the seed, not in the soil.

The vast majority of embryos produced in vitro and transferred fail to develop into an infant, supporting the concept that only a small fraction of embryos is destined to become a live birth. One of the main reasons for such a low embryo-to-infant ratio is the remarkably high number of embryos that after preimplantation genetic diagnosis are found to have a chromosome imbalance. This study reports the overall biological wastage from oocytes inseminated to ongoing pregnancies in patients undergoing preimplantation genetic screening (PGS) because of advanced age, recurrent pregnancy losses or multiple failed IVF cycles. The analysis of biological wastage per oocyte showed that in this cohort of patients, of 333 eggs inseminated, 183 (55.0%) provided embryos for biopsy, and of these, only 33 (18.0% per embryo and 9.9% per oocyte) were normal. A total of 26 embryos were suitable for transfer (14% per embryo and 7.8% per oocyte), but only five (1.5%) implanted and three (1.0%) resulted in live births. In conclusion, there is enormous biological wastage during assisted reproduction, and the data obtained from both embryos and oocytes of patients undergoing PGS support the concept that many embryos and eggs obtained during IVF are intrinsically abnormal and thus fail to implant.

Selective insulin-like growth factor-I antagonist inhibits mouse embryo development in a dose-dependent manner

To study the role of a synthetic insulin-like growth factor-I receptor (IGF-IR) antagonist, picropodophyllin, for mouse preimplantation embryo development in vivo and in vitro. In vitro and in vivo study. Hospital-based research unit. FVB/N mice and mouse embryos. The effect of picropodophyllin in mouse embryo development in vivo and in vitro, immunohistochemistry, ELISA, polymerase chain reaction. Embryo development, presence of IGF-IR, messenger RNA expression, IGF-I synthesis. The effect of picropodophyllin on embryo development in vitro and in vivo was not reversible. Mice treated with picropodophyllin 1 to 3 days after mating had a reduced number of blastocysts, 40.5% versus 78.8%, and a higher number of embryos with delayed development, 48.6% versus 11.5%. Insulin-like growth factor-IR protein is present in both phosphorylated and nonphosphorylated form at all stages of embryo development. The relative IGF-IR messenger RNA expression was highest in the oocyte and reduced during development to blastocyst stage. Insulin-like growth factor-I in culture media was reduced after picropodophyllin treatment. We conclude that IGF-I has an important role in normal mouse embryo development and that its receptor plays an essential role in the embryonic genome activation process.

61 MITOCHONDRIA DISTRIBUTION IN FERTILIZED, PARTHENOGENETIC, AND CLONED BOVINE EMBRYOS

In this study we evaluated mitochondrial distribution of individual bovine embryos after IVF, parthenogenetic activation (PG), and somatic cell nuclear transfer (SCNT). COCs obtained from slaughterhouse ovaries were matured in vitro for at least 18 h in TCM-199 supplemented with hormones, and then divided into 3 groups. SCNT and PG oocytes were stripped by vortexing in HEPES-HECM (hamster embryo culture medium) medium (HH) containing hyaluronidase and metaphase II (MII) oocytes selected by visualization of a polar body. The PG group oocytes were exposed 24h post-maturation to 5 μM ionomycin in HH for 4 min, then rinsed 3 times in HH and allocated to a 4-h culture in 2 mM DMAP in KSOM for activation. The SCNT group oocytes were included in a nuclear transfer procedure performed as previously described (Ross et al. 2006 Biotechniques 41, 741-750). Activation was performed as described for the PG group. The IVF group COCs were co-incubated for 20 h with 106 spermatozoa/mL in IVF-TALP supplemented with heparin. To label mitochondria, 1 mM MitoTracker CMXRos Red (Molecular Probes, Eugene, OR, USA) was added to HH at a final concentration of 0.3 μM. Samples were cultured for 15 min, washed in HH, placed in a glass-bottomed 35-mm Petri dish, and then observed and live photographed by using a spinning disk confocal microscope (Nikon Eclipse TE2000-E + CARV Confocal) equipped with a Cascade 512 B camera (Roper Scientific, Tucson, AZ, USA) using a Nikon 40×, 1.3 NA oil objective lens. Z series images were taken acquiring 15 focal planes at 10-μm intervals. Analysis was performed using Metamorph software. Samples were taken at pronuclear, 4 cell, and morula stages. Each sample was classified for its mitochondrial localization in pericytoplasm, cytoplasm, and perinuclear. Data was analyzed by proc glimmix (SAS, Cary, NC, USA). Significance was set at P &lt; 0.05. A similar pericytoplasmic distribution of mitochondria for all treatments up to the 4-cell stage was observed. At the pronucelar stage, mitochondria distribution was mostly pericytoplasmic, changing to cytoplasmic at the 4-cell stage. At the morula stage there was a significantly higher number of embryos with perinuclear distribution in IVF than in PG and SCNT embryos (Table 1). Our findings demonstrate that mitochondrial reorganization differs in fertilized more-developed embryos compared with their activated counterparts. This may have implications for further embryo development, mainly after SCNT. Table 1.Mitochondria distribution in fertilized, parthenogenetic, and cloned bovine embryos

Biology of the subtropical sac-spawning euphausiid Nyctiphanes simplex in the northwestern seas of Mexico: Vertical and horizontal distribution patterns and seasonal variability of brood size

Abstract Vertical and horizontal distributions of the subtropical euphausiid juvenile and adult Nyctiphanes simplex were mapped from samples collected during winter and summer 2007 in the Gulf of California, Mexico. During winter, wide-ranging high densities occurred in most of the Gulf of California. Densities decreased considerably during summer, with only at few locations having high densities. N. simplex made short daily vertical migrations of 50 m, clearly avoiding layers with temperatures >20 °C. In both seasons, N. simplex occurred above the low-oxygen layer ( −1 ), which occurred at 150 m during winter and 90 m during summer. In summer, this layer extended farther north and into shallower water columns than during winter. The low-oxygen layer acts as the bottom limit of vertical distribution and horizontal distribution is limited at the southern part of the gulf to temperatures >23 °C. Seasonal brood size and reproductive effort were estimated for both sides of the Baja California Peninsula under ship board experiments as a proxy of the relative effect of seasonal environmental conditions for euphausiid reproduction. Experiments were done during March, July, and December 2004 at the entrance to Bahia Magdalena and its westward continental shelf and in November 2005 and January and July 2007 in the Gulf of California. Contrary to broadcast-spawning euphausiids, N. simplex , a sac-spawning euphausiid, has a significant association of the brood size as a function of the total length of females. N. simplex produces an average brood of 52 eggs female –1 (range 5–116 eggs female –1 ) with a estimated total fecundity of 936 eggs female –1 in a life span (360–1337 eggs female –1 ), of which about 8% of its carbon weight is released per spawn, significantly higher than estimates of previous studies. In Bahia Magdalena, broods contained more embryos in March and July 2004 than in December 2004 when temperatures increased to >23 °C. In the Gulf of California, broods had higher numbers of embryos in November and July than in January 2007, suggesting that N. simplex has an out-of-phase reproductive season on both coasts of the peninsula. Reproductive investment effort was larger in the Gulf of California than in Bahia Magdalena, where females generated up to 18.3% of their weight in their broods, primarily by brood sizes produced from females between 10–12 mm total length that were particularly fecund during November and July.

Embryo management for the moderate responder: high cryoaugmented pregnancy rates following pronuclear freezing for patients with five or six embryos

OBJECTIVE: Many IVF programs advocate blastocyst culture for patients with high numbers of embryos (≥7). The optimal management of moderate responders (5-6 embryos) is unknown. Embryo transfer is typically performed on day 3 and cryopreservation only occurs if surplus embryos continue to develop into blastocysts. Cryopreservation at the pronuclear stage (PN) should allow excess embryos to be stored as an effective means for preserving embryos' developmental potential. We report our experience with PN cryopreservation and fresh and cryoaugmented (fresh plus FET) clinical pregnancy rates (PR) for moderate responders.

Fossil embryos and adult Viviparidae from the Early–Middle Pleistocene of Gesher Benot Ya’aqov, Israel: ecology, longevity and fecundity

Ashkenazi, S., Klass, K., Mienis, H.K., Spiro, B. & Abel, R. 2010: Fossil embryos and adult Viviparidae from the Early–Middle Pleistocene of Gesher Benot Ya’aqov, Israel: ecology, longevity and fecundity. Lethaia, Vol. 43, pp. 116–127. A unique fossil assemblage of the extinct freshwater viviparids Viviparus apameae galileae and Bellamya sp. with intact embryos enabled the assessment of population ecology parameters applied so far only to recent living populations. The assemblage, from the Early Middle Pleistocene site of Gesher Benot Ya’aqov, occurs in the sedimentary sequence above the Matuyama Brunhes Boundary (MB boundary) at 0.78 Ma. This fossil assemblage is related to a shoreline habitat that was affected by desiccation during a sudden drop in water level. In both species, the youngest females with embryos had five or more whorls, with heights of >24.8 and >16.1 mm in Viviparus and Bellamya respectively. Comparison of shell height and width, aperture height and width and the ratio of shell height/width indicated that females are significantly longer and larger than the shells without embryos. Longevity was estimated as at least 9 years for Viviparus and 8 years for Bellamya and lifetime fecundity of female viviparids was estimated based on longevity and reproductive period. The high number of reproducing females and high number of embryos indicate favourable habitat and climate conditions for both Viviparidae. The fossil Viviparidae from GBY have the characteristics of organisms that are K-selected by natural selection forces. □Fecundity, fossil embryos, longevity, molluscs, Pleistocene, reproductive output, sexual dimorphism, viviparids.

Plant regeneration through somatic embryogenesis of yacón [smallanthus sonchifolius (poepp. and endl.) H. Robinson

Smallanthus sonchifolius has tuberous roots containing large amounts of fructo-oligosaccharides and its medicinal use has increased due to the hypoglycemic properties reported for this species. An efficient system for propagation via somatic embryogenesis is reported using petiole segments cultivated on MS medium supplemented with combinations of BA, kinetin and 2,4-D, under light and darkness conditions. Embryogenic callus was formed in most of the treatments; however, somatic embryogenesis was promoted by the presence of light. Clusters of somatic embryos appeared on callus surface after 50 days of culture. The highest number of embryos was produced on 0.45 µM BA and 4.5 µM 2,4-D. Embryogenic calli were maintained on MS medium containing 4.5 µM BA and 0.045 µM 2,4-D. Embryos converted on hormone-free half-strength MS medium with 2 g.L-1 activated charcoal and plantlets were transferred to non-sterile conditions for acclimatization, showing 100% of survival.

Ovulation, Pregnancy Rate and Early Embryonic Development in Vernal Transitional Mares Treated with Equine- or Porcine-FSH

The objective of this study was to compare the efficacy of purified equine- and porcine-FSH treatment regimes in mares in early vernal transition. Mares (n = 22) kept under ambient light were examined ultrasonographically per-rectum, starting January 30th. They were assigned to one of two treatment groups using a sequential alternating treatment design when a follicle >or= 25 mm was detected. In the eFSH group, mares were treated twice daily with equine-FSH, and in the pFSH group mares were treated twice daily with porcine-FSH; treatments were continued until follicle(s) >or= 35 mm, and 24 h later hCG was administered. Oestrous mares were inseminated with fresh semen and examined for pregnancy on days 11-20 post-ovulation. In the eFSH group, 11/11 (100%) mares developed follicle(s) >or= 35 mm, 8/11 (73%) ovulated and 6/8 (75%) conceived. In the pFSH group, 5/11 (45%) developed follicle(s) >or= 35 mm, 4/11 (36%) ovulated and 3/4 (75%) conceived. Treatment with eFSH resulted in a greater ovarian stimulation; higher number of pre-ovulatory-sized follicles, higher number of ovulations and higher number of embryos (p < 0.05). Following ovulation, serum progesterone concentrations were correlated with the number of CLs and supported early embryonic development; maternal recognition of pregnancy occurred in all pregnant mares. We concluded that eFSH can be used to effectively induce follicular growth and ovulation in vernal transitional mares; however, if bred, diagnosis and management of twins' pregnancies would be required prior to day 16 because of the increased risk of multiple embryos per pregnancy. Conversely, the current pFSH treatment regime cannot be recommended.

Optimization of somatic embryogenesis and selection regimes for particle bombardment of friable embryogenic callus and somatic cotyledons of cassava ( Manihot esculenta Crantz)

Although a number of transformation systems and selection regimes have been developed for cassava (Manihot esculenta Crantz), they have only been applied in a limited number of genotypes. This limitation of the applicability of the systems is due largely to variation in morphological responses of different genotypes to the regeneration and transformation procedures employed, which underscores the need to study all possible parameters for any given cultivar. Using two cultivars (“Rosinha” and “Buja Preta”) we made an attempt to improve the frequency of somatic embryogenesis (SE), establish friable embryogenic callus (FEC) lines from developing somatic embryos and evaluate the effect of different concentrations of the antibiotics kanamycin, on secondary somatic embryogenesis (SSE) and of paromomycin, on FEC in proliferation and histodifferentiation media. Further, we report on transient expression of the visual marker gene uidA following particle bombardment of FEC from ‘Buja Preta’ and of cut pieces of green somatic cotyledons from ‘Rosinha’, using the plasmid pBI426. Higher number of embryos, which emerged as early as 8 days after culture on MS medium (CIM), was obtained when abaxial part of the explant was in direct contact with the medium as against the adaxial side, and this did not depend on the explant size (0.5 cm² or 2.5 cm²). Highly friable and embryogenic callus was obtained on GD medium supplemented with 2% (w/v) sucrose and 12 mg/L picloram. While paromomycin, at concentration of 60 mg/L arrested the proliferation and histodifferentiation of FEC, kanamycin in CIM containing explants undergoing SSE led to a decrease in their embryogenic potential resulting in tissue death at 50 mg/L. The highest transient expression of uidA gene in FEC was observed combining plasmolysis, M5 particle and a helium pressure of 1,200 psi, while in somatic cotyledons, the highest expression was observed when M5 particle was used along with the helium gas pressure of 900 psi.

Optimization of somatic embryogenesis and selection regimes for particle bombardment of friable embryogenic callus and somatic cotyledons of cassava (Manihot esculenta Crantz)

Although a number of transformation systems and selection regimes have been developed for cassava (Manihot esculenta Crantz), they have only been applied in a limited number of genotypes. This limitation of the applicability of the systems is due largely to variation in morphological responses of different genotypes to the regeneration and transformation procedures employed, which underscores the need to study all possible parameters for any given cultivar. Using two cultivars (“Rosinha” and “Buja Preta”) we made an attempt to improve the frequency of somatic embryogenesis (SE), establish friable embryogenic callus (FEC) lines from developing somatic embryos and evaluate the effect of different concentrations of the antibiotics kanamycin, on secondary somatic embryogenesis (SSE) and of paromomycin, on FEC in proliferation and histodifferentiation media. Further, we report on transient expression of the visual marker gene uidA following particle bombardment of FEC from ‘Buja Preta’ and of cut pieces of green somatic cotyledons from ‘Rosinha’, using the plasmid pBI426. Higher number of embryos, which emerged as early as 8 days after culture on MS medium (CIM), was obtained when abaxial part of the explant was in direct contact with the medium as against the adaxial side, and this did not depend on the explant size (0.5 cm² or 2.5 cm²). Highly friable and embryogenic callus was obtained on GD medium supplemented with 2% (w/v) sucrose and 12 mg/L picloram. While paromomycin, at concentration of 60 mg/L arrested the proliferation and histodifferentiation of FEC, kanamycin in CIM containing explants undergoing SSE led to a decrease in their embryogenic potential resulting in tissue death at 50 mg/L. The highest transient expression of uidA gene in FEC was observed combining plasmolysis, M5 particle and a helium pressure of 1,200 psi, while in somatic cotyledons, the highest expression was observed when M5 particle was used along with the helium gas pressure of 900 psi.

Assisted reproductive technology in Latin America: an example of regional cooperation and development

BACKGROUND: Since 1990, the Latin American Registry of Assisted Reproduction covers more than 80% of the assisted reproductive technology (ART) cycles performed regionally. METHODS: 130 centers enter their data online, and after collection, it is analyzed and published regionally. RESULTS: This paper analyses trends in ART procedures initiated between 1990 and 2004, and babies born up to 2005. Overall, the data include 150,000 embryo transfer (ET) cycles, 33,500 deliveries and the birth of 44,978 babies. The yearly increment in initiated cycles is below other regions of the world. The economic, religious and social factors explaining this limited access to ART are discussed. Major trends include: an increase in the age of female partner undergoing treatments; a marked shift towards an increase in the use of ICSI; and a steady increase in the delivery rates per ET reaching 24.6% in IVF/ICSI cycles, and 30.7% in OD cycles. Despite a slight decrease in the mean number of ET (2.9), almost half of all births are either twins or triplets. CONCLUSIONS: Our region is confronted with two main issues: limited access to those who can afford it; and a high number of embryos transferred resulting in almost 50% of babies born from multiple births.

Plant regeneration via indirect somatic embryogenesis and optimisation of genetic transformation in Coffea arabica L. cvs. Caturra and Catuaí

A protocol for Coffea arabica L. cvs. Caturra and Catuai plant regeneration via indirect somatic embryogenesis (ISE) was established. Furthermore, a biolistic mediated genetic transformation protocol was optimized for Catuai callus aggregates. Maximum callus induction was obtained when Caturra (87%) and Catuai (67%) leaves were cultured on Murashige and Skoog medium with 18.56 µM kinetin and 4.52 µM2,4-dichlorophenoxyacetic acid (2,4-D). Catuai suspension cultures were established from embryogenic callus using liquid proliferation CP and Sli media and diffused light and darkness. The higher suspension cultures fresh weight was obtained using Erlenmeyer (1425.4 ± 354.9 mg) than Recipient for Automated Temporary Immersion System (RITA ® ) (518.6 ± 55.1 mg), whereas the dry weight of suspension cultures was not significantly affected by the culture system used. Higher number of embryos per vessel (307.6 ± 49.0) and their fresh weight (9.6 ± 1.5 mg) were obtained with semisolid R medium than S3 medium. The highest somatic embryo development (25.0 ± 2.7) and fresh weight (780.0 ± 85.4 mg) were obtained with 1 min of immersion every 8 hrs. Higher fresh weight of regenerated plantlets was obtained with liquid Yasuda medium in RITA® (124.6 ± 16.3 mg) than semisolid media (36.3 ± 11.3 mg). For genetic transformation, the effect of helium pressure (900 and 1550 psi), and target distance (9 and 12 cm) and plasmid (pCAMBIA 1301, pCAMBIA 1305.2 and pCAMBIA 1301-BAR) on transient uidA expression Catuai suspension cultures were evaluated. The highest number of blue spots was obtained using 900 psi and 9 cm (125.8 ± 17.3). Stable uidA expression was observed on Catuai callus aggregates transformed with pCAMBIA 2301 and cultured on 100 mg l -1 of kanamycin.

Favorable influence of local injury to the endometrium in intracytoplasmic sperm injection patients with high-order implantation failure

Local injury to the endometrium prior to controlled ovarian stimulation may considerably improve implantation rates and pregnancy outcomes in intracytoplasmic sperm injection patients with high-order implantation failure (> or =4 IVF trials and > or =12 transferred embryos).

Effects of B chromosomes and supernumerary segments on morphometric traits and adult fitness components in the grasshopper, Dichroplus elongatus (Acrididae)

The South American species, Dichroplus elongatus, is polymorphic for B chromosomes and supernumerary segments in chromosome pairs M6 (SS6), S9 (SS9) and S10 (SS10). Both forms of supernumerary heterochromatin shape chiasma frequency and distribution and B chromosomes also affect male fertility. Here, we analysed the effects of these polymorphisms on morphometric traits (total, 3rd femur, 3rd tibia, thorax and tegmen lengths) and several adult fitness components, including male and female mating success, and female reproductive potential. B chromosomes tend to decrease, and SS6 segments to increase the body size of carriers. The analysis of reproductive potential suggested that B chromosome carrying females have higher numbers of embryos per clutch and ovarioles per ovary. The uni- and multivariate analysis of mating success revealed that sexual selection favours larger individuals of both sexes and males with standard karyotype. B chromosomes may have accumulation mechanisms, which involve preferential transmission of B chromosomes to germ cells or functional gametes. The maintenance of Bs might be explained by interactions among accumulation mechanisms and trade-offs between detrimental and favourable effects on different fitness components.

Survival of mouse embryos after vitrification depending on the cooling rate of the cryoprotectant solution

The aim of the study was to determine the relationship between the rate of cooling of eight-cell mouse embryos to the temperature of liquid nitrogen (-196 degrees C) and their developmental capacity after thawing on the basis of their ability to leave the zona pellucida ('hatching') during in vitro culturing. Eight-cell embryos were obtained from superovulated female mice and divided into three experimental and one control group. Embryos from the experimental groups were cryopreserved by the vitrification method using ethylene glycol as cryoprotectant. The vitrification protocols used in the study differed in the rate of cooling of the cryoprotectant solution. Embryos from the first group were frozen in conventional 0.25-ml plastic straws, those from the second group in pipetting 'tips', and embryos from the third group, placed in vitrification solution, were introduced dropwise directly into liquid nitrogen. The control group of embryos was cultured in vitro without freezing in a culturing medium in an environment consisting of 95% air and 5% CO2. The developmental capacity of thawed embryos was assessed on the basis of their ability to leave the zona pellucida ('hatching') after three days of in vitro culturing. In the control group 95.1% of embryos 'hatched'. A significantly higher number of embryos that 'hatched' after thawing was observed in the group introduced dropwise directly into liquid nitrogen (60.0%) compared to the group frozen in pipetting 'tips' (37.9%). The group frozen in straws yielded significantly the lowest proportion of 'hatching' embryos (8.1%). These results showed that increasing cooling rates during vitrification of embryos improved their survival.