Microsurgery on bovine embryos at the morula stage to produce monozygotic twin calves

Mature Brangus donor cows were superovulated with follicle stim-ulating hormone administered twice daily in intramuscular injections. On day 6.5 to 7 post-estrus, embryos were collected non-surgically using a phosphate-buffered saline medium. A total of 37 ova was collected, of which 28 were advanced morulae and early blastocysts. Twenty of these embryos were selected for micromanipulation with a radial-type Leitz micromanipulator. While the embryos were in a holding medium containing 10% fetal calf serum, three glass microinstruments were used to open the zona pellucida, remove the mass of blastomeres and bisect the embryo on a vertical plane. Halved embryos were inserted into bovine zonae and placed either as single half-embryos or twin half-embryos in 0.25 ml French straws with fresh holding medium. The micromanipulated embryos (demi-embryos) were then non-surgically transplanted, either as a single demi-embryo or as a twin demi-embryo pair, into the uterine horn of day 6.5 to 8 recipient beef females ipsilateral to the existing corpus luteum. Of the 14 micromanipulated embryos that were transplanted to recipients, pregnancy rates were 16.6% for the single demi-embryos and 62.5% for the twin demi-embryos. No pregnancies resulted from bisected blastocysts.     

Production of solasodine by Solanum laciniatum using plant tissue culture technique

Leaf and hypocotyl explants of 15 days old aseptically grown seedlings of Solanum laciniatum were cultured on MS medium supplemented with NAA (2 mg/l) and kinetin (0.5 mg/l) for callus initiation. For maintenance and proliferation of callus MS medium supplemented with 2,4-D (1 mg/l) and kinetin (0.5 mg/l) was used. The growth of the calli derived from hypocotyls increased with time of incubation and remained almost constant after 45 days. The solasodine content in callus culture was maximum after 30 days of incubation. Addition of L-arginine in the medium (50-150 mg/l) increased growth as well as chlorophyll content in the callus culture. The solasodine content also increased up to 1.2 to 1.4 times in these cultures. High frequency shoot regeneration was obtained in MS medium having BA (4 mg/l) and IBA (0.25 mg/l). For shoot multiplication, MS medium having BA (4 mg/l) was used. Shoots rooted on the same medium. Organogenesis promoted solasodine accumulation in the cultures. Regenerated shoots yielded higher solasodine content than undifferentiated as well as organogenic callus. Solasodine contents in the regenerated shoots was found to be 10 times higher than the callus culture and approached towards the field grown plants. Thin layer chromatography revealed the presence of three compounds. The most predominant spot (Rf 0.789) corresponded to the reference solasodine.     

Production of crocin using Crocus sativus callus by two-stage culture system

Saffron callus was grown in a two-stage culture on B5 medium supplemented with casein hydrolysate (300 mg l^–1) at 22 °C in dark with naphthalene acetic acid (2 mg l^–1) and 6-benzyladenine (1 mg l^–1) to give maximum biomass (16 g dry wt l^–1), and with indole 3-acetic acid (2 mg l^–1) and 6-benzyladenine (0.5 mg l^–1) for crocin formation. The maximum crocin production (0.43 g l^–1) was achieved by this two-stage culture method, which was three times that by a one-stage method.     

Production of infectious Epstein-Barr virus by using an intermittent culture collection system.

Procedures were developed to produce infectious Epstein-Barr virus in large glass fermentors with limited mechanical agitation by using an intermittent culture collection system. The system appears quite suitable for use in fermentation vessels of 50 liters or larger.     

Human placental cells in culture: a panning technique using a trophoblast-specific monoclonal antibody for cell separation

A monoclonal antibody specific to human trophoblast has been used to separate these cells from a mixture of heterogeneous cell types following trypsinization of placental villi. A panning technique was used whereby antibody-labelled cells were incubated on a goat-anti-mouse-IgG-coated surface. The adhered cells were kept in culture for several days and assessed using three different monoclonal antibodies as cell markers. These cultures were shown to be enriched in trophoblast cells.     

Epigenetic variation during the adult lifespan: cross-sectional and longitudinal data on monozygotic twin pairs

The accumulation of epigenetic changes was proposed to contribute to the age-related increase in the risk of most common diseases. In this study on 230 monozygotic twin pairs (MZ pairs), aged 18-89 years, we investigated the occurrence of epigenetic changes over the adult lifespan. Using mass spectrometry, we investigated variation in global (LINE1) DNA methylation and in DNA methylation at INS, KCNQ1OT1, IGF2, GNASAS, ABCA1, LEP, and CRH, candidate loci for common diseases. Except for KCNQ1OT1, interindividual variation in locus-specific DNA methylation was larger in old individuals than in young individuals, ranging from 1.2-fold larger at ABCA1 (P = 0.010) to 1.6-fold larger at INS (P = 3.7 × 10(-07) ). Similarly, there was more within-MZ-pair discordance in old as compared with young MZ pairs, except for GNASAS, ranging from an 8% increase in discordance each decade at CRH (P = 8.9 × 10(-06) ) to a 16% increase each decade at LEP (P = 2.0 × 10(-08) ). Still, old MZ pairs with strikingly similar DNA methylation were also observed at these loci. After 10-year follow-up in elderly twins, the variation in DNA methylation showed a similar pattern of change as observed cross-sectionally. The age-related increase in methylation variation was generally attributable to unique environmental factors, except for CRH, for which familial factors may play a more important role. In conclusion, sustained epigenetic differences arise from early adulthood to old age and contribute to an increasing discordance of MZ twins during aging.     

Production of Bacillus thuringiensis spores in total cell retention culture and two-stage continuous culture using an internal ceramic filter system

The production of Bacillus thuringiensis spores was investigated in a bioreactor incorporating a ceramic membrane filter to improve spore concentration and volumetric productivity. Two cultivation methods were used in this study: a total cell retention culture (TCRC), and a two-stage continuous culture with partial cell bleeding. In the TCRC, fed by 50 g/L of glucose, a spore concentration of 1.6 x 10(10) CFU/mL was obtained with a spore percentage of greater than 95% and a maximum cell mass of 82.2 g/L. The volumetric productivity was four times higher than that obtained from batch cultivation. In the two-stage continuous culture with partial cell bleeding spore concentration was strongly dependent on the bleed ratio. The spore concentration of 1.8 x 10(9) CFU/mL and the spore percentage of 70% were obtained at the second stage when a bleed ratio of 0.33 and a dilution rate of 0.23 h(-1) were used. (c) 1993 John Wiley & Sons, Inc.     

Obesity-related derangements of coagulation and fibrinolysis: a study of obesity-discordant monozygotic twin pairs

Coagulation and fibrinolytic activities are under strong genetic control. We studied the effects of acquired obesity, independent of genetic factors on coagulation and fibrinolysis activities in obesity-discordant healthy monozygotic (MZ) twin pairs. Fourteen obesity-discordant (BMI within-pair difference >3 kg/m(2)) and 10 concordant (BMI difference <2 kg/m(2)) MZ twin pairs were identified from the nationwide FinnTwin16 study. Body composition (dual-energy x-ray absorptiometry), abdominal fat distribution (magnetic resonance imaging), liver fat (magnetic resonance spectroscopy), high sensitivity C-reactive protein, insulin sensitivity (euglycemic hyperinsulinemic clamp), and a panel of different markers of blood coagulation and fibrinolysis in the fasting state were measured. Strong resemblance was observed in most coagulation factors within all twin pairs, with the intraclass correlations ranging from 0.73 to 0.97, P < 0.03. However, the activities of fibrinogen and FIX, FXI, and FXII, and plasminogen activator inhibitor-1 (PAI-1) activities were increased in the obese co-twins (P < 0.05) and strongly correlated with the measures of adiposity, inflammation, and insulin resistance (r = 0.32-0.73, P < 0.05) among the twin individuals. Intrapair differences in fibrinogen and PAI-1 correlated with those in BMI, adiposity, and fasting insulin levels (r = 0.40-0.58, P < 0.05) indicating the independent effect of obesity. Derangements of blood coagulation and fibrinolysis are present already in early adulthood in obese subjects. Acquired obesity, independent of genetic factors, increases the activities of fibrinogen and activities of FIX, FXI, FXII, and PAI-1. This study confirms the mechanisms of simultaneous activities of intrinsic coagulation factors and impaired fibrinolysis predisposing obese subjects to thrombosis.     

New method for culture of zona-included or zona-free embryos: the Well of the Well (WOW) system

Culture of mammalian zygotes individually and in small groups results in lower developmental rates than culture of large groups. Zona-free zygotes also have impaired developmental potential in current culture systems. This paper describes a new approach to resolve the problems, the Well of the Well (WOW) system. Small wells (WOWs) were formed in four-well dishes by melting the bottom with heated steel rods. The WOWs were then rinsed, the wells were filled with medium, and the embryos were placed into the WOWs. To test the value of the WOW system a 3 x 3 factorial experiment was performed. Bovine presumptive zygotes were cultured from day 1 to day 7 (day 0: day of insemination) using three modules (single embryos, embryo groups of five, or single zona-digested embryos) and three different culture systems (400 microl medium, 200 microl drops, or WOWs). An additional control group consisted of 40 to 50 embryos cultured in 400 microl medium. The WOW system resulted in higher blastocyst/oocyte rates for all three modules (single: 59%; group of five: 61%; single zona-digested: 53%) than the culture in drops or in wells (P < 0.05 for all). The developmental rate was independent of the number of WOWs per well. The cell number of blastocysts cultured in the WOW system did not differ from that of the controls. Apart from its theoretical value in revealing the role of different factors influencing embryo development in vitro, the WOW system may have immediate practical consequences in certain areas of mammalian embryo production.     

The variability of ovum pick-up response and in vitro embryo production from monozygotic twin cows

Oocytes were recovered by ovum pick up (OPU) from nine pairs of monozygotic twin German Simmental cows. The hypothesis was that there is less variability between identical twins versus among non-related individuals in the variation in the recovery of oocytes by OPU and in the efficiency of in vitro embryo production. Estrous cycles were synchronized with two doses of cloprostenol, 11 days apart. Beginning 3-4 days after synchronized estrus, OPU was done twice weekly (every 3 or 4 days; total of 11 sessions). The influence of repeated OPU on estrous cyclicity was established by estrus detection, plasma progesterone concentrations, and ovarian ultrasonography. There were no differences among days of collection for the number and quality of cumulus oocyte-complexes (COCs), and rates of cleavage and blastocyst formation. A total of 1,661 COCs, including 657 (39.6%) good-quality COCs, were recovered. From 1,457 (87.7%) cultured COCs, 827 zygotes cleaved and 314 blastocysts were produced on Day 7. The total number of COCs and the blastocyst rates varied among pairs of monozygotic twins; within pairs, only slight differences were observed. In conclusion, recovery of COCs and production of embryos had substantially less variation within pairs of monozygotic twins than among non-related cattle.     

Multiple shoot culture of Dianthus caryophyllus using mist culture system

Summary A mist bioreactor system for the plant tissue cultures was developed. Using this system, the growth of Dianthus caryophyllus multiple shoots was directly measured. Tissue growth in mist bioreactor system was far better than that on agar medium and almost comparable to that in liquid medium. The mass increase (final dry weight/initial dry weight) in the mist culture was 2.85 while 3.28 in the liquid flask culture. Shoots were seriously vitrified in flask culture but these vitrifications could be considerably cured by using the mist culture system.     

Investigating unique environmental contributions to the neural representation of written words: a monozygotic twin study

The visual word form area (VWFA) is a region of left inferior occipitotemporal cortex that is critically involved in visual word recognition. Previous studies have investigated whether and how experience shapes the functional characteristics of VWFA by comparing neural response magnitude in response to words and nonwords. Conflicting results have been obtained, however, perhaps because response magnitude can be influenced by other factors such as attention. In this study, we measured neural activity in monozygotic twins, using functional magnetic resonance imaging. This allowed us to quantify differences in unique environmental contributions to neural activation evoked by words, pseudowords, consonant strings, and false fonts in the VWFA and striate cortex. The results demonstrate significantly greater effects of unique environment in the word and pseudoword conditions compared to the consonant string and false font conditions both in VWFA and in left striate cortex. These findings provide direct evidence for environmental contributions to the neural architecture for reading, and suggest that learning phonology and/or orthographic patterns plays the biggest role in shaping that architecture.     

Development of in vitro fertilized embryos of the polyclad flatworm,Hoploplana inquilina,following blastomere separation and deletion

Summary After in vitro fertilization of naked eggs of the polyclad turbellarian, Hoploplana inquilina, both cell separation experiments and deletions of specific blastomeres are possible. With these techniques one can analyze the developmental potential of isolated blastomeres and determine if the embryonic axes have been established at the four-cell stage in this primitive, equally-cleaving spiralian embryo. Two-cell separation experiments with development of both halves resulted in pairs of larvae 1) neither of which had an eye (29%), 2) both of which had one eye (19%), and 3) one of which was eyeless and the other was one-eyed (43%). Deletion of one blastomere at the four-cell stage resulted in 68% one-eyed, 28% two-eyed and 3% eyeless larvae. The one-eyed larvae were asymmetric with respect to eye position with more having right than left eyes. Abnormal or missing ventrolateral lobes occurred with deletion of any of the macromeres at four cells but were significantly more common when A or C rather than B or D was deleted. The experiments support the hypothesis that eye development is a consequence of cytoplasmic localization of both a specific eye precursor and an inducer which segregate independently of cleavage planes, and indicate that the embryonic axes have been determined at the four-cell stage.     

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

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

Using monozygotic twin differences to examine the relationship between parental affection and personality: a life history account

The relationship between maternal and paternal affection, reported in adulthood, and personality was examined using a genetically sensitive research design comparing differences between monozygotic twins. Using life history theory as a framework, it was predicted that differences in maternal and paternal affection would be predictive of differences in personality such that the twin reporting greater maternal and paternal affection would also report a personality profile reflective of a slow life history strategy. Specifically, it was predicted that the twin that reported greater maternal and paternal affection would also score high on the meta-traits of plasticity, stability, and the general factor of personality (GFP). The results supported the hypotheses, with most variance accounted for by the GFP. Additional results suggest that differences in paternal affection exhibit a stronger effect and that stability and plasticity may provide unique information about the association between differences in parental affection and differences in personality. Attachment and parental investment theories offer possible explanations for the findings, although alternative explanations are also proffered. It may also be beneficial for future research using a monozygotic twin difference approach to utilize biometric measures of life history strategy.     

The cause of the shorter gestation length for twin calves: A computer simulation

The possibility that the shorter length of gestation for twin calves is because pregnancy is determined by the earlier maturing twin has been investigated by computer simulation. The simulation indicated that this explanation alone could account for only about half the shortening of gestation for natural twin calves, and it is suggested that the combined actions of both twins is superimposed on the stimulus of the earlier maturing twin to initiate parturition. With mixed-species twins produced by embryo transfer, the immaturity of the more slowly maturing Bos indicus is such that it cannot cooperate with the earlier maturing Bos taurus cotwin to shorten gestation to appreciably less than is normal for single Bos taurus pregancies.