Sequential uterine horn versus simultaneous total uterine flush to recover bovine embryos nonsurgically

Beef cows were superovulated with follicle stimulating hormone (FSH) to compare two nonsurgical methods of embryo recovery from the uterus. In the first method each uterine horn was independently flushed with physiological saline solution (PSS) through a Foley catheter passed through the cervix and into the uterine horn. In the second method both uterine horns were simultaneously flushed with PSS by passing the catheter into the uterine body. In both methods, the numbers of ovulations were determined after embryo collection by counting the corpora lutea (CL) on both ovaries of each cow through a flank incision. Independent flushing (n = 19) averaged 6.4 embryos and 16.1 CL per cow for a recovery rate of 40%. Simultaneous flushings (n = 22) averaged 5.4 embryos and 17.7 CL per cow for a recovery rate of 31%. This difference between the recovery rates of the two flushing methods was not significant (P>0.05).     

A single cannula technique for nonsurgical collection of ova from cattle.

The uteri of 34 heifers were flushed for ova six to nine days following estrus using a single cannula nonsurgical technique. The technique involved the infusion of fluid by gravity and agitation within the uterus by to-and-fro action of a syringe followed by unassisted fluid collection. Each horn was flushed five times using 30–150 ml of flushing fluid per flush. Recovered fluid (flushing fluid plus uterine secretion) was an average of 95% of the volume of the fluid inserted. Ova were recovered from 12 of 19 nontreated, single ovulating heifers (63%) and from all of 15 superovulated heifers (mean and S.D. for number of ova, 6.3 ± 4.4/ superovulated heifer; range, 1 to 14 ova). Based on the number of corpora lutea, the ova recovery index was 54% as averaged over the 15 superovulated heifers. The technique has been used in 4 additional superovulated heifers with modification (increased number of flushes to 8) subsequent to the termination of the planned project. Recovery index for the first 5 flushes was 58%. However, some ova were recovered in the 6th, 7th, and 8th flushes resulting in an apparent improved recovery index of 69%.     

Distribution of spermatozoa in the female reproductive tract of the domestic cat in relation to ovulation induced by natural mating

The purposes of this study were to demonstrate the localization of spermatozoa in the reproductive tract of female domestic cats before (30 min and 3 h after mating) and after ovulation (48 and 96 h after mating), and to evaluate the efficiency of two techniques for studying sperm distribution. Estrus was induced in twenty-four female cats using 100 IU eCG and the females were divided into four groups with six females per group. The same male cat was used for mating with all the females. One group of six females was mated once; the others were mated four times in 1 h. Ovariohysterectomy was performed at 30 min, 3 h, 48 h, and 96 h after mating and the excised reproductive tracts were divided into seven segments on each side: infundibulum, ampulla, isthmus, uterotubal junction (UTJ), cranial and caudal uterine horn, and uterine body. The vagina and the lumina of the segments from one side were flushed with 0.5 ml PBS. The flushed and the non-flushed segments from the contralateral side were then fixed in 3% neutral buffered formalin and processed for routine histology. The numbers of spermatozoa in the flushings and in 40 histological sections from each segment were counted. Before ovulation, the majority of spermatozoa was detected in the vagina and the uterine segments, whereas after ovulation, significantly higher numbers of spermatozoa were present in the uterine tubal segments. The decreasing gradient in sperm numbers at 30 min and 3 h after mating between the vagina, the uterine segments, including the UTJ, and the uterine tubal segments indicated that the cervix and the UTJ served as barriers for sperm transport in the cat. The UTJ and the uterine crypts acted as sperm reservoirs before ovulation whereas the isthmus was a sperm reservoir around the time of ovulation. There was no difference in sperm numbers in the tissue sections between flushed and non-flushed segments, implying that the flushing technique only recovered some intraluminal spermatozoa while most of the spermatozoa remained in the epithelial crypts. This was further supported by the finding that significantly higher numbers of spermatozoa were recovered in the flushings at 30 min and 3 h after mating, when more spermatozoa were free in the lumina, than at 48 and 96 h after mating, when the majority of the spermatozoa were entrapped in the uterine epithelial crypts.     

Improvement in recovery of embryos/ova using a shallow uterine horn flushing technique in superovulated Holstein heifers

The aim of this study was two-fold: (1). to compare recovery of embryos/ova from superovulated Holstein heifers by flushing the uterine horns through insertion of the catheter very close to the tip of the horn (deep) or just after the uterine bifurcation (shallow) and (2). to evaluate the hormonal and superovulatory response to estradiol benzoate (EB) treatment prior to superovulation. Ten Holstein heifers (12-16 months) underwent two superovulatory treatments in a cross-over design. Heifers were treated with decreasing doses of FSH from Days 8 to 12.5 of a synchronized estrous cycle. At 4 days prior to superovulation, half of the heifers received EB (5mg, i.m.) or served as Controls, followed by the alternative treatment in the subsequent superovulation. At embryo recovery, one uterine horn was flushed with deep ( approximately 7 cm caudal to the tip of the horn) and the other with shallow ( approximately 5 cm cranial to the beginning of the uterine bifurcation) flushing techniques. Embryos/ova were recovered, counted, and scored. Number of ovulations was estimated by ultrasound. Pretreatment with EB reduced circulating FSH and regressed the first wave dominant follicle with no change in number of large follicles, number of ovulations, number of embryos/ova recovered, or number of transferable embryos. The shallow flushing technique was superior to the deep technique for number of embryos/ova recovered per horn (5.4+/-1.1 versus 3.9+/-0.8) or percentage of embryos/ova recovered per CL (63.9+/-8.6% versus 37.4+/-6.5%). Thus, flushing the entire uterine horn increased recovery of embryos/ova.     

Uteroglobin and the accumulation of progesterone in the uterine lumen of the rabbit

Summary This study was undertaken to determine whether the influx of progesterone into the uterine lumen of the rabbit, in the preimplantation period, is dependent onuteroglobin (UGL). Rabbits were ovariectomized and, three months later, treated with two defferent doses of progesterone. Purified UGL was injected into one uterine horn and, as a control,immunoglobulin G (IgG) was injected into the other. After four days, the animals were sacrificed their uteri flushed, and the progesterone content of the washes was determined by radioimmunoassay.Animals with the lower serum progesterone level (2.8 ng/ml) had a significantly different uterine horn progesterone content. The hormone accumulation in the horn containing UGL was 2.3 to 7.5 times higher than in the horn containing IgG. Animals with a higher serum progesterone level (7.2 ng/ml) showed no differences. The hormone content was equally high in both horns, presumably due to the synthesis of endogenous UGL being reactivated by the hormone treatment.The validity of these experiments as models for the events during early pregnancy and the physiological role of progesterone available inside the uterus are discussed.     

Effect of prostaglandin F(2)alpha at insemination on sperm cell numbers and pregnancy rate in beef cattle

Fourteen cycling, nonlactating, multiparous beef cows were artificially inseminated (AI) 10 to 12 h after the onset of natural estrus. One unit of frozen-thawed semen containing 100 x 10(6) total sperm cells was deposited into the body of the uterus. Immediately after AI, alternating cows were injected i.m. with either 25 mg (5 ml) of prostaglandin F(2)alpha (PGF) or 5 ml of 0.9% saline-benzyl alcohol control solution. Cows were slaughtered 16 +/- 1 h post AI, oviducts were retrieved, segmented into thirds (upper, middle and lower) and flushed with 1 ml of 0.2% gluteraldehyde in phosphate buffered saline. The number of sperm cells was counted using a phase contrast microscope. There were no right or left side effects (P=0.61) on the number of sperm cells recovered per oviduct within cow (389 vs 553; average SEM = 219). PGF had no effect (P=0.77) on the number of sperm cells recovered per oviduct (642 vs 300; average SEM = 231 for PGF and control females, respectively). More sperm cells were recovered from the lower third segment (P<0.05) compared with the middle and upper segments. Ovulation tended to affect (P=0.10) the number of sperm cells recovered per oviduct (742 vs 200; average SEM = 231). Additionally, 114 beef females (68 Angus x Hereford heifers and 46 Chianina crossbred postpartum suckled cows) were treated as described above following AI at natural estrus with 20 x 10(6) motile sperm cells. Pregnancy rates did not differ significantly in heifers (70.6 vs 58.8%) or in Chianina cows (34.8 vs 52.2%) for control and PGF-treated females, respectively. Overall, pregnancy rates were identical between control and PGF-treated females at 56.1%. In this study, PGF treatment immediately following AI in beef cattle had no effect on the number of sperm cells in the oviducts or on the pregnancy rate.     

Column chromatography and electrophoresis of bovine uterine lumenal proteins collected during the estrous cycle

Uteri of 29 normally cycling Holstein and Jersey cows were non-surgically flushed with 50 ml sterile 1.5% saline, and fluids were recovered to evaluate biochemical methods for determination of qualitative changes in uterine lumenal protein at known stages of the estrous cycle. Total protein (mg) and number of red blood cells (million/ml) were 17.7 and 9.8; 7.6 and 7.1; 9.1 and 6.0; and 26.2 and 2.8 at day 0, 5, 10 and 15 of the bovine estrous cycle. Column chromatography (Sephacryl S-200) of uterine secretions revealed seven uterine specific peaks at ambient temperatures. One peak may be a hemoglobin contaminant. Five uterine specific protein peaks representing proteins greater than 160 000, ～ 25 000 and less than 13 700 mol. wt. (3) were eluted with high performance liquid chromatography. Native polyacrylamide disc gel electrophoresis fractionated uterine fluid into as many as 13 bands. There were differences in six protein bands between uterine fluid and plasma. The consistency between Sephacryl S-200 and high performance liquid chromatography is the presence of three to four low molecular weight (< 13 700) uterine specific proteins. Sephacryl S-200 chromatography resulted in elucidation of a uterine specific protein approximately 60 000 mol. wt. not found with high performance liquid chromatography. However, proteins with mol. wt. > 160 000 and approximately 25 000 were found with high performance liquid chromatography. Results indicate no differences in protein class during the estrous cycle and that red blood cell contamination must be monitored during qualitative evaluation of uterine proteins.     

Superovulatory response of river bufalo (Bubalus bubalis )

The ovarian response of 25 buffalo-cows was visually assessed, and their oviducts and uteri separately flushed 3 to 6 d post superovulatory estrus at slaughter. Ten buffalo-cows slaughtered on Days 5 and 6 were examined per rectum for corpora lutea (CL) and follicles > 8 mm prior to slaughter, and the estimate was compared later with the actual ovarian response. Five out of the ten buffalo-cows were nonsurgically flushed in vivo on Day 5 of the estrous cycle, a day before slaughtering, and as a result, six ova/embryos were recovered. After the flushing of the reproductive tract at slaughter, one more ovum was recovered from the uterus of each of the three buffalo-cows. As a result of treatment of three groups of five buffalo with 3000 IU pregnant mare serum gonadotrophin (PMSG) on Days 6, 10 or 14 of the estrous cycle, 3.8, 6.2 and 3.4 CL on the average were recovered, respectively (Experiment I). A mean number of 8.8 and 9.0 CL, respectively, was obtained in two groups of five buffalo each, after treatment with 40 mg of follicle stimulating hormone (FSH) on Day 10 of the stage of the estrous cycle (Experiment II) and 3000 IU PMSG regardless of the stage of cycle (Experiment III). The percentage of ova/embryos recovered in the three experiments was 32.8, 20.4 and 22.2, respectively.     

Maintenance of milk protein gene expression in a subpopulation of 7,12-dimethylbenz (a) anthracene-induced rat mammary carcinoma cells grown on attached collagen gels

Summary Milk protein gene expression was studied in cell subpopulations of 7,12-dimethylbenz(a)anthracene-induced rat mammary carcinoma cells enriched or depleted for casein production grown on attached collagen gels. Culture of these cells in the presence of 10% fetal bovine serum, insulin (5 μg/ml), hydrocortisone (10 μg/ml), and prolactin (5 μg/ml) maintained α-, β-, and γ-casein and whey acidic protein mRNAs at levels identical to cells isolated from perphenazine-treated rats. Whey acidic protein mRNA levels in the tumor cells relative to the 14-d lactating gland were greater than those of the casein mRNAs. Withdrawal of prolactin from the casein-producing cells resulted in the loss of all four milk protein mRNAs. Subsequent addition of prolactin to the withdrawn cells caused a rapid accumulation of these mRNAs to prewithdrawal levels. Milk protein gene expression in this tumor cell subpopulation is modulated by prolactin (in the presence of insulin and hydrocortisone) in a similar manner to that observed in the normal mammary gland when these tumor cells are cultured on attached collagen gels. This work was supported by National Institutes of Health grant CA 16303. M. L. Johnson was the recipient of NIH Fellowship, HD 06157.     

A replacement for methoxyflurane (Metofane) in open-circuit anaesthesia

Methoxyflurane (Metofane) has been widely used as an open-circuit anaesthetic in small laboratory animals for several decades. Its low vapour pressure and high blood solubility have permitted its use in convenient and simple drop-chamber/nose-cone setups. Recently, following the decision by the primary manufacturer to discontinue production, it has become increasingly difficult to obtain methoxyflurane. We describe here a simple and effective adaptation of isoflurane, an excellent inhalation anaesthetic, to open-circuit drop-chamber/nose-cone anaesthesia. It was found that the vapour concentration of isoflurane could be continuously varied by dissolving the anaesthetic in propylene glycol and that a 20% solution produced effective anaesthesia such that in adult mice, 2 ml of 20% isoflurane in propylene glycol induced anaesthesia within 2 min in a one-litre drop chamber. Furthermore, anaesthesia maintenance with 20% isoflurane was tested in two sets of mice. In one set, surgical plane anaesthesia was maintained for 10 min in a head chamber. After removal of the chamber, the animals awoke within one minute and recovered without any indication of post-anaesthetic distress. The second set contained pregnant mice; here anaesthesia was maintained for between 10 and 12 min, during which laparotomy, exposure of one uterine horn, intrauterine injection and wound closure were completed. The recovery from anaesthesia was also within a minute and with no signs of distress. Healthy litters were delivered after a normal gestation. This isoflurane/propylene glycol procedure is simple, effective and humane, and is a good substitute for methoxyflurane.     

Nonsurgical uterine stage preimplantation embryo collection from the common marmoset

A nonsurgical technique for the recovery of uterine stage preimplantation embryos was developed for the common marmoset (Callithrix jacchus). In 54 flush attempts, using 19 different animals, 54 morphologically normal embryos, seven unfertilized oocytes or degenerate embryos, and five empty zonae pellucidae were recovered, giving a recovery rate of 1.0 embryo per flush or 1.2 ovulation products per flush. Because the ovarian cycles of common marmosets can be synchronized with prostaglandin PGF2α, multiple marmosets can be flushed in a short period, providing age-matched embryos for controlled experiments.     

Role of prostaglandins in development of porcine blastocysts

Rapid elongation of porcine blastocysts between Days 11 to 12 of pregnancy coincides with an increase in uterine luminal content of prostaglandins. The present study evaluated the effect of two prostaglandin synthesis inhibitors (indomethacin and flunixin meglumine) on elongation of porcine blastocysts from spherical to filamentous forms between Day 11 to 12 of pregnancy. Gilts were hemi-hysterectomized on Day 11 of prenancy. The excised uterine horn was flushed with 0.9% saline and diameter of blastocysts recovered were measured. Immediately following surgery, pregnant gilts were assigned to receive either: 1) vehicle every 4 h, 2) flunixin meglumine (banamine) every 4 h, or 3) indomethacin every 12 h. The remaining uterine horn was removed and flushed after the time of blastocyst elongation estimated for each gilt on basis of blastocyst development in the first horn. Uterine flushings were analyzed for total calcium, protein, acid phosphatase activity, estrone, estradiol-17β and prostaglandin F. Pretreatment blastocyst diameter was similar for all groups and ranged from 1 mm to 20 mm. Treatment of gilts with either banamine or indomethacin effectively inhibited (P<0.001) the increase in uterine luminal content of PGF. Total calcium, estrone and estradiol-17β were not influenced by treatment. Total uterine luminal protein and acid phosphatase activity were reduced (P<0.05) in banamine treated gilts compared to those receiving vehicle or indomethacin treatments. Although total PGF recovered in uterine flushings was reduced during the period of blastocyst elongation, treatment with PGF synthetase inhibitors failed to block rapid elongation of blastocysts from the spherical to filamentous forms.     

Transuterine transport of spermatozoa after artificial insemination in heifers

Eight heifers were artificially inseminated with frozen-thawed semen during heat. Semen was deposited in one of the uterine horns. The animals were slaughtered 2 h after insemination and the genital tract was flushed. Sperm concentration in the flushing fluid was estimated by haemocytometric counting.There was a considerable transport of spermatozoa from the site of semen deposition to the uterine horn and oviduct on the opposite side. Spermatozoa were recovered from all parts of the oviduct (infundibulum, ampulla and isthmus) and distal and proximal parts of the horn on the non-inseminated side. In 7 out of 8 heifers more spermatozoa were recovered from the side of the tract opposite to insemination than from the inseminated side, although the differences were small in 2 animals. No clear relationship could be seen between ovarian activity and distribution of spermatozoa.     

Stimulation of alkalothermophilic Aspergillus terreusxylanase by low-intensity laser radiation

In this study, Aspergillus terreus was irradiated by a 7.3 mW He–Ne laser in the presence of crystal violet, toluidine blue O and hematoporphyrin as photosensitizers. Xylanases recovered from non-irradiated and irradiated fungi were purified and characterized. The maximum production of xylanase (42.2 U/ml) was obtained after 5 min of laser irradiation in the absence of the photosensitizer. The irradiation of the sensitized fungus diminished the production of xylanase. On purification using G-100, the specific activity of xylanase recovered from the irradiated fungus was 292 U/mg protein representing a 37-fold purification over the crude extract compared with 95.6 U/mg protein representing the 12.8-fold for the enzyme recovered from the non-irradiated fungus. The enzyme recovered from the irradiated fungus had lower molecular weight as compared with that recovered from the non-irradiated one. Characterization of the purified enzymes revealed that the enzyme recovered from the irradiated fungus was more thermostable and had a wider range of optimum reaction temperature (60–70°C) and pH (4.0–12.0), compared to the non-irradiated one.     

Survival of DNA-injected cow embryos temporarily cultured in rabbit oviducts

Holstein or Angus cows were superovulated, inseminated with fresh bull semen, and necropsied about 12 h after estimated time of ovulation. Ova were centrifuged at 15,600 G for 3 to 8 min to reveal pronuclei. In Experiment 1, pronuclear bovine embryos were transferred to ligated or unligated oviducts of 1-d pseudopregnant rabbits for 7 d; 30 of 32 embryos were recovered from ligated oviducts but only 2 of 26 from oviducts and uterine horns of unligated oviducts. In Experiment 2, a Rous sarcoma virus-chloramphenicol acetyl transferase fusion gene was injected into one pronucleus of about half of 404 fertilized bovine ova, using a micromanipulator and interference contrast optics. Injected and noninjected embryos were then transferred to opposite ligated rabbit oviducts. Embryos were recovered after 7, 8 or 9 d. Of 120 centrifuged but ininjected embryos recovered from rabbit oviducts, 66 (55%) were in the morula to hatching blastocyst stage of development. Of 105 embryos centrifuged and injected with foreign DNA, 55 (52%) were in the morula to hatching blastocyst stage. In Experiment 3, centrifuged bovine embryos, noninjected or DNA-injected, were cultured in rabbit oviducts for 7 d then transferred nonsurgically to the uterus of recipient cows. Embryos were also flushed from superovulated cows 8 d after estrus and transferred directly to recipient cows. After 7 d, the uterus of recipient cows was flushed nonsurgically to recover embryos. The proportion of transferred embryos recovered with normally elongated trophoblastic membranes and the proportion of recipient cows with developing embryos were 14 of 25 DNA-injected embryos, 5 of 8 cows; 6 of 15 centrifuged but noninjected embryos, 4 of 6 cows; and 11 of 29 embryos transferred directly, 5 of 8 cows. Results indicate that bovine embryos can be cultured in rabbit oviducts and survive after transfer to cow uteri and that injection of foreign DNA may not increase embryonic loss within the first 2 wk after injection.     

Callus regeneration from cotyledon protoplasts ofChamaecyparis obtusa (Hinoki cypress)

Summary Protoplasts were isolated from cotyledons of 1- to 1.5-mo.-old seedlings ofChamaecyparis obtusa using 1% driselase or 0.25% pectolyase Y-23 in combination with 1% cellulase RS in 0.6M mannitol solution. Cell division and colony formation were induced efficiently in liquid Murashige and Skoog’s (MS) medium containing 0.6M mannitol and 10 to 30 μM 2,4-dichlorophenoxyacetic acid or 1 μM of naphthaleneacegic acid at the cell density of 1 to 2×10³ ml. Continued callus proliferations was observed by transferring tissue to fresh medium of the same composition as the induction medium without mannitol. Campbell and Durzan’s medium and ammonium nitrate-free MS medium were less effective than MS medium. High concentration of benzyladenine (1 or 10 μM) was inhibitory to cell division.     

Assessment of a new utero-tubal junction insemination device in dairy cattle

A new artificial insemination device for semen deposition near the utero-tubal junction in cattle (Ghent device) has been developed at the Ghent University (Belgium). In this study, the effect of the new insemination device on sperm quality was evaluated. Moreover, in a field trial 4064 dairy cows were inseminated by 12 inseminators to examine the efficacy of the device under field conditions.The Ghent device is a disposable plastic catheter which can easily follow the curvature of the uterine horns and thus reach the utero-tubal junction (UTJ). After expulsion of the inseminate with 0.7 or 1.7 ml of air, 19.0% of the insemination dose remained in the insemination catheter. Sperm loss can be diminished to 9.0% of the original insemination dose when the insemination catheter is flushed with 0.1 ml of air, followed by 0.6 ml of physiological saline solution. No toxic effect of the insemination catheter on sperm quality or fertilizing capacity was found. In the field trial, sperm were inseminated in dairy cattle which were divided in three groups. The first group was inseminated in the uterine body with the conventional insemination device, the second group in the uterine body with the Ghent device, and the third group in the tip of both uterine horns with the Ghent device. Each insemination was performed with 10 x 10(6) to 15 x 10(6) frozen-thawed spermatozoa. The pregnancy rates (PRs) were significantly affected by the insemination technique (P = 0.02), by the inseminator (P = 0.01), by heifer or cow (P < 0.01), and by the insemination number (P < 0.01). Pregnancy rates obtained with the conventional insemination device (57.6%) were significantly better than those obtained with the Ghent device in the uterine body (52.7%) (P < 0.01), but did not differ significantly from those obtained after deep insemination into both uterine horns (53.8%) (P = 0.27). It can be concluded that the Ghent device is suitable for utero-tubal junction insemination of dairy cattle under field conditions. Whether the Ghent device is also suitable for insemination with lower insemination doses is at present under investigation.     

Prolonged mating in prairie voles (Microtus ochrogaster) increases likelihood of ovulation and embryo number

Prairie voles are induced ovulators that mate frequently in brief bouts over a period of approximately 24 h. We examined 1) impact of mating duration on ovulation and embryo number, 2) incidence of fertilization, 3) temporal pattern of embryo development, 4) embryo progression through the reproductive tract over time, and 5) embryo development in culture. Mating was videotaped to determine first copulation, and the ovaries were examined and the reproductive tracts flushed at 6, 8, 10, 12, 16, 20, and 24 h and 2, 3, and 4 days after first copulation. The number of mature follicles and fresh corpora lutea and the number and developmental stage of embryos were quantified. One, two-, and four-cell embryos were cultured in Whitten's medium. Mature follicles were present at the earliest time examined (6 h). Thirty-eight percent of females that had been paired for < 12 h after the first copulation ovulated, whereas all females paired >/= 12 h after the first copulation ovulated. Virtually all (> 99%) oocytes recovered from females paired for >/= 12 h after first copulation were fertilized. Pairing time after first copulation and mean copulation-bout duration were significant (p < 0.05) determinants of embryo number. Embryos entered the uterine horns and implanted on Days 3 and 4, respectively, after first copulation (Day 0). Embryos cultured in vitro underwent approximately one cell division per day, a rate similar to that in vivo. We conclude that prairie voles ovulate reliably after pairing for >/= 12 h, although some females showed exceptional sensitivity not predicted by the variables quantified. Prolonged mating for longer than 12 h increased the total embryos produced. This mechanism likely has adaptive significance for increasing offspring number.     

Primary and long term epithelial cell cultures from human fetal normal colonic mucosa

Summary Primary and passaged cultures of normal colon epithelial cells, derived from human fetuses (13 to 17 wk of conceptual age) have been established. These cultures have been passaged 16 times thus far. The cultures have been initiated and maintained in medium consisting of 50% Dulbecco's minimum essential medium and 50% Ham's F12 medium and supplemented with antibiotics (penicillin, 100 U/ml; streptomycin, 100 μg/ml); ascorbic acid, 40 μg/ml;l-isoleucine, 50 μg/ml; epidermal growth factor, 20 ng/ml; insulin, 5 μg/ml; cholera toxin, 5 ng/ml; transferrin, 1 μg/ml; fetal bovine serum (10%); and HEPES, 25 mM final concentration, and incubated at 37°C in humidified gas containing 5% CO₂: 95% air. The cellular and subcellular characteristics of primary and passaged cultures were defined using light microscopy and scanning and transmission electron microscopy. The cells exhibited microvilli on cell surfaces and showed junctional complexes and interdigitations between cells. Indented nuclei with dense chromatin and marginated heterochromatin, numerous mitochondria, rough endoplasmic reticulum, polysomes, and extensive Golgi zones were conspicuous. Also, periodic acid Schiff's reagent-positive staining of the cells suggests the active synthesis of complex mucopolysaccharides in the cytoplasm. This study was supported by USPHS Grant CA-30185 from the National Large Bowel Cancer Project, National Cancer Institute.     

Sieve size effects on root length and biomass measurements of maize (Zea mays) and Grevillea robusta

The purpose of this study was to investigate the effects of different mesh sizes on the recovery of root length and biomass and to determine whether the degree of recovery was influenced by plant species and sample location. Sieves of 2.0, 1.0, 0.5 and 0.25 mm (4.0, 1.0, 0.25 and 0.06 mm2) mesh sizes were used to recover and measure the root length and biomass of Zea mays L. (maize) at 0–15 cm and 30–45 cm depths and of Grevillea robusta A. Cunn. ex R. Br. (grevillea) at the same depths 1.0 m and 4.5 m from a line of grevillea trees. At 0–15 cm, the coarser sieves (sum collected with 2.0 and 1.0 mm sieves) recovered approximately 80% of the total root biomass measured, but only 60% of the root length. The proportion of total maize root length and biomass recovered by the coarser sieves decreased with soil depth. The proportion of total grevillea root length recovered by the coarser sieves was similar at the two soil depths, but increased slightly with distance from the tree line. The ≥ 0.5 mm sieves recovered between 93 and 96% of grevillea and maize root biomass and between 73 and 98% of their root length, depending on the sample location. Roots passing through the 0.5 mm sieve, but recovered by the 0.25 mm sieve were about 20% of total maize root length and grevillea root length at 1.0 m from the tree line but < 5% of the total grevillea root length at 4.5 m from the tree. Roots passing through the 0.5 mm sieve but recovered by the 0.25 mm sieve contributed only slightly to root biomass. Although the ≥ 0.5 mm sieves provided adequate measurements of root biomass, the ≥ 0.25 mm sieves were required for accurate measurement of fine root length. There was no universal correction for root length and biomass underestimation when large sieve sizes were used because the proportions of length and biomass recovered depended on the plant species and on soil depth and distance from the plant. This revised version was published online in August 2006 with corrections to the Cover Date.