Comparative 2-DE proteomic analysis of date palm (Phoenix dactylifera L.) somatic and zygotic embryos

Two-dimensional gel electrophoresis coupled to mass spectrometry has been used to compare the proteome of date palm (Phoenix dactylifera L. cv. Deglet Nour) zygotic and somatic embryos. Proteins were trichloroacetic acid–acetone–phenol extracted, quantified, and resolved by 2-DE in the 5 to 8 pH range. Total protein content and number of resolved spots were higher in zygotic (110 ± 14.5 mg/g DW; 349 spots) than in somatic (70.96 ± 4.8 mg/g DW; 210 spots) embryos. The 2-DE map of both systems showed qualitative (263) and quantitative (72) differences. Statistical analysis of spot intensity was performed by PCA, obtaining two accurate groupings of the samples and determining the most discriminating spots. Samples were also clustered using Euclidean distance with average linkage algorithm of the Genesis software package. Sixty-three variable spots were subjected to mass spectrometry analysis, resulting in 23 identifications. Identified proteins were classified in the following functional categories; glycolysis (8 proteins), citrate cycle (1), ATP synthesis (1), carbohydrate biosynthesis (2), amino acids metabolism (1), stress related (4), storage (3), and with no function assigned for three of them. Most of the somatic embryo specific proteins identified belonged to glycolysis pathways, whereas those of the zygotic embryo to storage and stress-related proteins. Differences are discussed in terms of metabolism and biology of both types of embryos.     

Somatic embryogenesis in the medicinal legume <Emphasis Type="Italic">Desmodium motorium</Emphasis> (Houtt.) Merr.

An efficient protocol was established for regeneration of Desmodium motorium via somatic embryogenesis. Embryogenic calli were induced from cotyledon segments (6 mm, 16 days old) lacking embryo axis, excised from seedlings grown in vitro on Murashige and Skoog (MS) medium supplemented with indole-3-acetic acid (IAA) (2.9 μM) in combination with 6-benzyladenine (BA) (4.44 and 8.88 μM). Differentiation of embryogenic calli into globular and heart-shaped somatic embryos was achieved on transfer to hormone-free MS medium. When incubated for 4 days on MS medium supplemented with BA (8.88 μM), 95% of the globular and heart-shaped somatic embryos matured into torpedo and cotyledonary stages with minimum (10%) abnormalities. Modified MS basal medium without hormones and containing half-strength macronutrients and 0.88 M sucrose was suitable for germination of mature somatic embryos. Regenerated plantlets were successfully transferred to earthen pots with survival rate of 50%. Secondary embryogenesis was observed when pre-existing somatic embryos at globular and heart-shaped stages were cultured on MS medium supplemented with various concentrations of BA, adenine sulphate (AdS) and abscisic acid (ABA) individually.     

Bottlenecks in Pinus radiata somatic embryogenesis: improving maturation and germination

Commercial deployment of clonal trees via somatic embryogenesis (SE) could increase forest productivity over conventional tree breeding techniques. However, some technical advances need to be made to use SE in clonal forestry with Pinus radiata. For example, the conversion of embryonal mass (EM) into plants is at present a major bottleneck. For this reason, maturation experiments were carried out to determine the effect of the initial amount of EM, activated charcoal (AC) and the best combination of abscisic acid (ABA), sucrose and amino acid concentration in the maturation medium. Germination was evaluated on different media formulations with and without AC. When 100 mg of EM were suspended in liquid medium without AC, cotyledonary somatic embryos were obtained in all the maturation media tested. Maturation medium supplemented with 60 μM ABA, 6% sucrose, and embryo development medium amino acid mixture produced the highest number of cotyledonary somatic embryos, between 10 and 1,550 embryos per gram of EM fresh weight. Approximately half of the tested 25 lines produced more than 600 embryos per gFW. Embryo development was the best when somatic embryos were germinated in half strength modified Quoirin and Lepoivre medium supplemented with 2 g L⁻¹ AC. This protocol simplified and improved SE maturation and germination due to the elimination of subcultures, the large number of somatic embryos obtained from a very low amount of EM, and the elimination of pre-germination treatments, resulting in a significant saving of cost and labor.     

In vitro production of bovine embryos in medium supplemented with a serum replacer: Effects on blastocyst development,cryotolerance and survival to term

In this study, we evaluated a serum replacer (SR; Knockout SR^®, Invitrogen) in our in vitro culture systems. We hypothesized that SR would benefit bovine embryo development, since SR supported survival of embryonic stem cells (which originate from embryos). Experiment 1 compared oocyte maturation with SR versus fetal bovine serum (FBS). Following fertilization, blastocyst development was lower for oocytes matured with SR (21.5 versus 34.1, P < 0.05). Experiment 2 evaluated SR for culturing embryos. Following fertilization, embryos were cultured for 3 days in KSOM, and then assigned to treatments: (1) KSOM static culture (KNM); (2) fresh KSOM (KD3); (3) KSOM + SR or (4) KSOM + FBS and cultured to Day 7 (fertilization = Day 0). Blastocyst development in FBS or SR was higher than either KNM or KD3 (48.2, 47.2, 32.7, and 35.5, respectively, P < 0.05). Experiment 3 evaluated cryosurvival of embryos cultured in the same manner as Experiment 2. On Day 7, embryos were vitrified and upon warming, embryos cultured in SR had greater 24 h survival rates (70.6%) than all other treatments (P < 0.05). Finally, Experiment 4 evaluated effects of SR on pregnancy rate and development to term. Culture in SR was not detrimental to pregnancy or calving rates (50 and 50%, respectively), and SR calves had normal birth weights (mean = 38.8 kg ± 1.5). In conclusion, the use of SR for maturation of oocytes was not beneficial; however, SR enhanced embryo culture by improving development in vitro, cryotolerance and survival, effectively replacing serum in culture.     

Long-term somatic embryogenesis and maturation of somatic embryos in Hevea brasiliensis

A high frequency of secondary embryogenesis was induced from isolated early cotyledonary-stage somatic embryos of Hevea brasiliensis. A long-term embryogenic line was established by the use of recurrent embryogenesis and maintained for 3 years on hormone-free medium by the transfer of selected proembryogenic masses every 10 days.

The addition of 234 mM sucrose as stress with sucrose and 10⁻⁵ M abscisic acid (ABA) to the culture medium enhanced the maturation of somatic embryos. Under these culture conditions, the embryo population was composed of 45% globular, 18% oblong and 37% torpedo-stage embryos. These somatic embryos had well-formed tissue structure, a well-defined epidermis, protein storage bodies, and a high accumulation of starch. The triglyceride content was five times as high in the torpedo-stage embryos that developed on medium supplemented with 234 mM sucrose and 10⁻⁵ M ABA as in embryos obtained on basal medium with 58 mM sucrose.     

Factors influencing somatic embryo maturation, high frequency germination and plantlet formation in Terminalia chebula Retz.

The factors influencing somatic embryo maturation, high frequency somatic embryo germination, and plantlet formation were studied in Terminalia chebula Retz. Maturation of somatic embryo were influenced by a number of factors such as in vitro culture passage, concentrations of sucrose, levels of abscisic acid (ABA), basal media and media additive combinations. Maximum frequency of somatic embryo maturation (57.22 ± 2.02), was obtained on MS medium supplemented with 50 g/l sucrose. Different factors such as strengths of MS nutrients, plant growth regulators, media additives and their combinations controlling somatic embryo germination and plantlet formation were studied. High frequency of germination and plantlet formation (58.80 ± 1.47) were achieved by subsequent subculture of mature somatic embryos on MS medium containing 30 g/l sucrose and 0.5 mg/l benzyladenine (BA). However, although duration of in vitro passage of the callus tissue was critical, contribution of the combinations of plant growth regulators and media additives showed nugatory effect on somatic embryo maturation and germination as evident from variable responses.     

One-step dilution of open-pulled-straw (OPS)-vitrified mouse blastocysts in sucrose-free medium

Embryos vitrified by the open-pulled-straw (OPS) method are only briefly exposed to cryoprotectants and not fully equilibrated with the cryoprotectant. That being the case, conceivably the post-thawing de- and rehydration processes may be omitted. This would render thawing and dilution in a single step and direct transfer to recipients possible without the need for a microscope and other laboratory equipment. Morphologically intact mouse blastocysts from superovulated 5- to 8-week-old virgin female NMRI mice were vitrified according to a protocol [6] slightly modified from the classical OPS-procedure of Vajta et al. [29] consisting of exposure to 10% dimethyl-sulfoxide (Me₂SO) + 10% ethylene glycol (EG) for 1 min, followed by 20% Me₂SO + 20% EG for 20 s before loading into straws that are plunged into liquid nitrogen. In Group 1, 75 blastocysts were exposed to the standard thawing and dilution regimen involving exposure to three solutions of decreasing sucrose content (Control). In Groups 2, 3 and 4, 75 blastocysts each were transferred, in a single step, to medium at 37 °C containing 0.66, 0.33 or 0 M sucrose, respectively. After 48 h of in vitro culture the proportion of hatched blastocysts was determined. In Group 1, this proportion amounted to 82.7%, in Groups 2, 3 and 4 to 76.0%, 73.3% and 78.7%, respectively (P > 0.05). To examine their potential to continue development in vivo, OPS-vitrified blastocysts thawed according to the regimens of Groups 1 and 4 were transferred to recipients (10 embryos/recipient). In Group 1, 9/10 recipients got pregnant with 4.7 ± 0.6 (mean ± SEM) fetuses, in Group 4, 8/10 recipients with 5.0 ± 0.5 fetuses. The overall embryo survival rate per group was 42% for Group 1 and 40% for Group 4. All fetuses were normally developed and viable and there were no significant differences between groups (P > 0.05). It may be concluded that warming and transfer of OPS-vitrified mouse embryos in a single step in medium devoid of sucrose is feasible, which is tantamount to a substantial simplification of embryo transfer operations.     

Effects of type of explant and age,plant growth regulators and medium strength on somatic embryogenesis and plant regeneration in <Emphasis Type="Italic">Eucalyptus camaldulensis</Emphasis>

Plant regeneration was achieved through direct and indirect somatic embryogenesis in Eucalyptus camaldulensis. Callus was induced from mature zygotic embryos and from cotyledon explants collected from 10, 15, 25, and 30-day-old seedlings cultured on Murashige and Skoog (MS) basal medium supplemented with different concentrations of naphthaleneacetic acid (NAA). Maximum callus induction from mature zygotic embryos was obtained on MS basal medium containing 1 mg l⁻¹ NAA. The frequency of callus development varied based on the age of the cotyledon explants 10-day-old explants giving highest percentage on MS basal medium supplemented with 1 mg l⁻¹ NAA. Callus obtained from mature zygotic embryos gave highest frequency of somatic embryogenesis on MS basal medium containing 0.5 mg l⁻¹ benzyladenine (BA) and 0.1 mg l⁻¹ NAA. Separate age wise culture of the calli, obtained from cotyledons of different ages cultured separately, revealed high somatic embryogenic potential on callus from 10-day-old cotyledons. Direct somatic embryogenesis too was obtained from hypocotyl explants without an intervening callus phase on MS basal medium containing 0.5 mg l⁻¹ BA. The effects of abscisic acid (ABA), sucrose, and different strengths of MS medium on somatic embryo maturation and germination were also investigated. Number of mature somatic embryos increased with lower concentrations (0–1 mg l⁻¹) of ABA while no significant differences were observed at higher concentrations (2–5 mg l⁻¹) of ABA. Compared to basal medium containing lower concentrations of sucrose (1%), the MS medium supplemented with higher levels of sucrose (4%) showed significantly lower frequency of mature somatic embryos. Basal medium without any dilution gave the highest number of immature embryos. However, the number of mature embryos was high at higher medium dilutions.     

Hatching,survival and deformities of piracanjuba (Brycon orbignyanus) embryos subjected to different cooling protocols

Groups of one hundred Brycon orbignyanus embryos at the stage of blastopore closure were subjected to different cooling protocols. Different combinations and concentrations of cryoprotectants were tested: sucrose, methanol, ethylene glycol and dimethyl sulfoxide (Me₂SO); at different temperatures (0.0 ± 2.0 °C and 8.0 ± 2.0 °C) and refrigeration times (6, 10, 24, 72 and 168 h), with the exception of the positive control (incubation without previous cooling). At the end of each refrigeration time, the embryos were acclimatized, rehydrated and incubated to determine hatching, survival and deformity rates. Morphological analysis of embryos was also carried out. The results showed that temperature and refrigeration time are critical factors for embryo survival. No embryos survived after 24, 72 and 168 h of refrigeration. Furthermore, when the refrigeration time increased from 6 to 10 h and the temperature decreased from 8.0 ± 2.0 °C to 0.0 ± 2.0 °C, mortality rates increased significantly. It was also found that in all protocols dead eggs and/or larvae with some degree of deformity were present. The main larval deformities observed were the malformation of the head, tail, yolk sac, vertebral column and eyes.     

Effect of transporting donor or recipient does and their embryos on the outcome of fresh embryo transfer in Boer goats

Between-farm embryo transfer of livestock animals can potentially increase the spread of quality genetic material. However, the transporting of donor or recipient animals or their embryos has become a practical problem. The objective of this study was to compare the effect of transporting donor and recipient does and their embryos between various farms on inter-farm fresh embryo transfer in Boer goats. Results indicate the transportation of donor does within 4 h before embryo collection not to have a significant effect on embryo recovery number, embryo survival rate and the subsequent pregnancy in recipient does. Also, the transportation of embryos at 36.5–38 °C within 2 h before embryo transfer did not significantly affect the embryo survival rate and subsequent pregnancy rate, but the transportation of embryos at 20 °C resulted in a significant (P < 0.05) lower survival rate (41.7%) and pregnancy rate (42.0%). The transportation of recipient does resulted in a significantly lower pregnancy rate (42.0%) and embryo survival rate (32.1%) than the transportation of donor does and embryos. Results suggest the transportation of donor does to be the best method for embryo transfer programs on the farm. Alternatively, the supply of fresh embryos kept at body temperature (36.5 °C) was also preferred for short or long distances between farms.     

Changes in protein profiles associated with somatic embryogenesis in peanut

The somatic embryogenesis potential of zygotic embryo axes of peanut (Arachis hypogaea L. cv. DRG-12) at different stages of development was evaluated by culturing on MS medium with 18.1 μM 2,4-dichlorophenoxyacetic acid (2,4-D). A 100 % frequency with 18.3 somatic embryos per explant was observed from 4 mm long immature zygotic embryo axes collected 31 – 40 d after pollination. Medium supplemented with 16.6 μM picloram resulted in slow development of somatic embryos whereas in the presence of 21.5 μM α-naphthaleneacetic acid (NAA), the explants underwent maturation with induction of roots after 30 d. The changes in protein profiles in zygotic embryo axes at different stages of development correlated with their potential to form somatic embryos. Immature zygotic embryo axes exhibited high frequency somatic embryogenesis in the stage preceding abundant accumulation of 22 and 65 kDa proteins. The content of 22 and 65 kDa proteins decreased immediately after culture on medium fortified with 18.1 μM 2,4-D and increased again after 12 d of culture coinciding with the development of somatic embryos on the explants. The content of 22 and 65 kDa proteins was low at 15 d of culture on medium supplemented with 16.6 μM picloram possibly due to slow development of the somatic embryos on the explant. On maturation medium containing 21.5 μM NAA, a marked increase in the content of 22 and 65 kDa proteins in 15 d-old cultures was observed.     

Influence of supplemental enzymes on the performance and phosphorus excretion of broilers fed wheat-based diets to 6 weeks of age

Efficacies of phosphorolytic enzymes (phytase+acid phosphatase), and an enzymic “cocktail” (phytase+acid phosphatase+pectinase+citric acid), were investigated in broilers fed wheat-based diets from day 1 to 43. Broilers were fed the following four diets: (1) a positive control diet (7.1 g total P/kg, 4.1 g non-phytate P/kg, 9.8 g Ca/kg); (2) a low phosphorus diet (4.1 g total P/kg, 1.7 g non-phytate P/kg, 6.0 g Ca/kg) supplemented with phytase (750 units/kg) and acid phosphatase (3156 units/kg); (3), a low phosphorus diet (4.1 g total P/kg, 1.7 g non-phytate P/kg, 6.0 g Ca/kg) supplemented with phytase, acid phosphatase, pectinase (1900 units/g) and citric acid (20 g/kg); and (4) a low phosphorus diet (4.1 g total P/kg, 1.7 g non-phytate P/kg, 8.0 g Ca/kg) supplemented as in diet 3. For the grower period (22–43 days), the contents of P and Ca were lowered by 0.2 and 0.3 g/kg, respectively. The dietary treatments were fed to three floor pen replicates of 50 birds each. For the starter period, there were no differences observed among dietary treatments in terms of body weight gains or feed efficiency. Total body weight gains for the starter and grower periods did not differ among dietary treatments, but total feed efficiency was significantly enhanced in birds fed diet 2. At the completion of the experiment chickens fed phosphorolytic enzymes had the best feed efficiencies, the highest contents of ash in the toes, and the highest carcass yield. Chicken receiving the cocktail of enzymes and 8 g Ca/kg (diet 4) performed as well as birds in the control treatment, but had higher yields of carcass and excreted 56% less phosphorus.     

Biological treatment of n-hexane and methanol in trickle bed air biofilters under acidic conditions

This study focuses on evaluating the degradation of n-hexane/methanol mixture in trickle-bed-air-biofilters (TBABs). Two different concentration ratios of methanol:n-hexane were evaluated (3:1) for TBAB “A” and (5:1) for TBAB “B”. Both TBABs were run and fed with nutrients buffered at pH 4 for encouraging the growth of fungi. The TBABs were loaded with pelletized diatomaceous earth support media and were run at an empty bed residence time of 120 s. n-Hexane loading rates (LRs) ranged from 0.9 to 13.2 g/m³ h for both TBABs. The corresponding methanol LRs varied from 2.3 to 37.7 g/m³ h and from 4.6 to 64.5 g/m³ h for TBABs “A” and “B”, respectively. Experimental results have shown that the degradation of n-hexane in presence of methanol is enhanced for n-hexane LRs less than 10.6 g/m³ h as compared to previous study for sole-fed n-hexane, but for n-hexane LRs of 13.2 g/m³ h, the performance of TBABs in eliminating n-hexane depended on the methanol to n-hexane ratios. The impact was less severe for TBAB “A” (RE 85%) as compared to TBAB “B” (RE 72%). This is attributed to the high LRs of methanol in TBAB “B”. n-Hexane performance stability was another advantage attained.     

Storage protein accumulation during zygotic and somatic embryo development in Picea abies (Norway spruce)

Total protein was extracted from zygotic embryos and from somatic embryos of Picea abies (L.) Karst. (Norway spruce) cultured in vitro at different times during their development. An analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 2-dimensional gel electrophoresis of the protein extracts showed that protein composition and the temporal changes in protein abundance were very similar in the two embryo types. Both zygotic and somatic embryos accumulated storage proteins in abundance during their maturation phase of growth; the somatic embryos when cultured on medium containing 90 m M sucrose and 7.6 μ M ABA. The major storage proteins are composed of polypeptides with molecular masses of about 22, 28, 33 and 42 kDa and they are identical in both embryo types according to their molecular mass and average isoelectric points. These proteins are also the most abundant proteins in the female gametophytic tissue of the mature seed.     

Multiple regeneration pathways in Spathoglottis plicata Blume — A study in vitro

Asymbiotic germination of immature seeds (embryos), and mature seeds and micropropagation of Spathoglottis plicata were described. Effects of three nutrition media namely, Murashige & Skoog (MS); Phytamax (PM); and Phyto-Technology orchid seed sowing medium (P₇₂₃), two carbon sources such as glucose and sucrose at 2–3% (w/v), two plant growth regulators such as 6-benzylaminopurine (BAP; 0.5–3.0 mg l^− 1) and α-naphthalene acetic acid (NAA; 0.5–2.0 mg l^− 1) and peptone (2.0 g l^− 1) were examined on seed germination, early protocorm development and micropropagation. The maximum germination of mature seeds (95%) was recorded in PM medium supplemented with 2% (w/v) sucrose + 2.0 g l^− 1 peptone. For germination of embryos P₇₂₃ medium supplemented with 1.0 mg l^− 1 BAP proved best. Multiple shoot buds or protocorm-like bodies (PLBs) were produced from stem segments of in vitro raised seedlings. Both direct organogenesis and embryogenesis were observed and the morphogenetic response was initiated by different concentrations and combinations of PGRs. The optimum PGR combination for maximal PLB regeneration was 1.0 mg l^− 1 NAA + 2.5 mg l^− 1 BAP, while 1.0 mg l^− 1 NAA + 1.0 mg l^− 1 BAP for shoot bud development. Strong and stout root system was induced in half strength PM medium supplemented with 0.5 mg l^− 1 IAA. The well-rooted plantlets were transferred to pots containing a potting mixture composed of saw dust, coconut coir, humus, and coal pieces at 1:1:1:2 (w/w) with 80% survival in outside environment and flowered after two years of transfer.     

Stress as adaptation? A test of the adaptive boost hypothesis among Batswana men

Many studies have found elevated cortisol linked to negative events (“stress”) and subsequent negative outcomes, such as reduced immunity and stunted growth, leading to the conclusion that high cortisol is “bad.” However, a growing number of studies have found more advantaged groups showing relatively elevated cortisol. For example, higher morning cortisol followed by a steeper diurnal decline among Caucasians compared to ethnic minorities has been interpreted as a context-specific “adaptive boost” to meet daily demands. We tested the adaptive boost hypothesis using data on socioeconomic status, depressive affect and salivary cortisol among adult men (n = 32) in Botswana. Three findings emerged: (i) depressive affect was associated with lower morning cortisol (r = ? 0.43, p = 0.014); (ii) depressive affect was associated with a diurnal increase in cortisol when comparing morning and evening samples (r = 0.49, p = 0.004); and (iii) depressive affect was associated with lower income (r = ? 0.55, p = 0.001). Findings are consistent with the adaptive boost hypothesis and add to a growing body of evidence that elevated cortisol is not universally bad. Hypothalamic–pituitary adrenal axis (HPAA) activity, such as cortisol, may be adaptive depending on a person's contextual circumstances. Based on our findings and those of previous studies, we develop a “person-in-context” model of the threat appraisal process. Integrated with life history theory, our model facilitates testable hypotheses about intra- and inter-individual variability in HPAA and adaptive consequences.     

The effect of inspiratory and expiratory loads on abdominal muscle activity during breathing in subjects “at risk” for the development of chronic obstructive pulmonary disease and healthy

The abdominal muscle activity has been shown to be variable in subjects with chronic obstructive pulmonary disease (COPD) when respiratory demand increases and their recruitment pattern may change the mechanics, as well as the work and cost of breathing. The scientific evidence in subjects “at risk” for the development of COPD may be important to understand the natural history of this disease. This study aims to evaluate the effect of inspiratory and expiratory loads on the abdominal muscle activity during breathing in subjects “at risk” for the development of COPD and healthy. Thirty-one volunteers, divided in “At Risk” for COPD (n = 17; 47.71 ± 5.11 years) and Healthy (n = 14; 48.21 ± 6.87 years) groups, breathed at the same rhythm without load and with 10% of the maximal inspiratory or expiratory pressures, in standing. Surface electromyography was performed to assess the activation intensity of rectus abdominis (RA), external oblique and transversus abdominis/internal oblique (TrA/IO) muscles, during inspiration and expiration. During inspiration, in “At Risk” for COPD group, RA muscle activation was higher with loaded expiration (p = 0.016); however, in Healthy group it was observed a higher activation of external oblique and TrA/IO muscles (p < 0.050). During expiration, while in “At Risk” for COPD group, RA muscle activation was higher with loaded inspiration (p = 0.009), in Healthy group TrA/IO muscle showed a higher activation (p = 0.025). Subjects “at risk” for the development of COPD seemed to have a specific recruitment of the superficial layer of ventrolateral abdominal wall for the mechanics of breathing.     

Selection of developmentally competent buffalo oocytes by brilliant cresyl blue staining before IVM

The brilliant cresyl blue (BCB) test determines the activity of glucose-6-phosphate dehydrogenase (G6PDH); the activity of this enzyme is greatest in growing oocytes, but it declines as oocytes mature. The objective was to develop and evaluate this test for assessing development of buffalo oocytes (to select developmentally competent oocytes for increased in vitro embryo production). Oocytes were exposed to BCB stain diluted in mDPBS (DPBS with 0.4% BSA) for 90 min at 38.5 °C in a humidified air atmosphere; those with or without blue coloration of the cytoplasm were designated as BCB+ and BCB−, respectively. In Experiment 1, oocytes were exposed to 13, 26, or 39 μM BCB. There were fewer BCB+ oocytes after exposure to 13 μM BCB (10%) than after exposure to 26 or 39 μM BCB (57.2 and 61.8%; P < 0.05), but there was no significant difference among treatments for blastocyst production rate. In Experiment 2, the diameter of BCB+ oocytes (144.4 ± 4.2 μm; mean ± S.E.M.) was higher (P < 0.05) than that of BCB− oocytes (136.8 ± 4.6 μm). In Experiment 3, oocytes were allocated into three groups: control (immediately cultured); holding-control (kept in mDPBS for 90 min before cultured); and treatment-incubation (incubated with 26 μM BCB). After IVM, oocytes were fertilized in vitro and cultured on an oviductal monolayer. The nuclear maturation rate was higher (P < 0.05) in BCB+ (86.2%), control (83.4%) and holding-control (82.6%) oocytes than BCB− (59.2%) oocytes. The BCB+ oocytes yielded more blastocysts than control or holding-control oocytes (33.4, 20.2, and 21.0%, P < 0.05); blastocyst development was lowest in BCB− oocytes (5.2%). In conclusion, staining of buffalo oocytes with BCB before IVM may be used to select developmentally competent oocytes for increased in vitro embryo production.     

Metabolism of terpenes in the response of grape (Vitis vinifera L.) leaf tissues to UV-B radiation

This study investigated the terpene profiles as determined by GC–EIMS analysis of in vitro cultured plants of Vitis vinifera exposed to a “field-like” dose of UV-B (4.75 kJ m⁻² d⁻¹) administered at two different fluence rates (low, 16 h at 8.25 μW cm⁻², and high 4 h at 33 μW cm⁻²). Low UV-B treatment increased levels of the membrane-related triterpenes sitosterol, stigmasterol and lupeol, more notable in young leaves, suggesting elicitation of a mechanism for grapevine acclimation. By contrast, accumulation of compounds with antioxidant properties, diterpenes α and γ tocopherol and phytol, the sesquiterpene E-nerolidol and the monoterpenes carene, α-pinene and terpinolene had maximum accumulation under high UV-B, which was accentuated in mature leaves. Also the levels of the sesquiterpenic stress-related hormone abscisic acid (ABA) increased under high UV-B, although 24 h post irradiation ABA concentrations decreased. Such increments of antioxidant terpenes along with ABA suggest elicitation of mechanism of defense. The adaptative responses induced by relatively low UV-B irradiations as suggested by synthesis of terpenes related with membrane stability correlated with augments in terpene synthase activity.