The role of the α7 subunit of the nicotinic acetylcholine receptor in the acute toxicosis of methyllycaconitine in mice.

The adverse physiological effects of methyllycaconitine (MLA) have been attributed to its competitive antagonism of nicotinic acetylcholine receptors (nAChRs). Recent research suggested a correlation between the lethal dose (LD50 ) of MLA and the amount of α7 nAChR in various mouse strains, suggesting that mice with more α7 nAChR require more MLA to be poisoned. The objective of this study was to characterize the role of the α7 subunit in the acute toxicosis of MLA by evaluating the acute toxicity of MLA in mice lacking the α7 subunit. The LD50 values for MLA were 4.2 ± 0.9, 3.7 ± 1.1 and 3.3 ± 0.9 mg kg(-1) body weight (BW) for wild-type, heterozygous knockout and homozygous knockout mice, respectively. We also evaluated the response of anabasine in these mice. The LD50 values for anabasine were 1.6 ± 0.3, 2.0 ± 0.4 and 1.8 ± 0.3 mg kg(-1) BW for wild-type, heterozygous knockout and homozygous knockout mice, respectively. The protein expresson of various nAChR subunits was compared to determine if mice lacking the α7 subunit compensate by over expressing other nAChR subunits. There were no significant differences in the protein expression of the α3 , α4 , α5 , ß2 and ß4 subunits amongst the three genotypes of mice in brain or skeletal muscle. The results of this study suggest that α7 nAChR does not play an integral role in the acute toxicosis of MLA or anabasine. Consequently other nAChR subunits of nAChRs found in the neuromuscular junction are probably the primary target for MLA and anabasine resulting in acute toxicosis.

The role of the α7 subunit of the nicotinic acetylcholine receptor on motor coordination in mice treated with methyllycaconitine and anabasine.

The adverse effects of methyllycaconitine (MLA) have been attributed to competitive antagonism of nicotinic acetylcholine receptors (nAChR). Research has indicated a correlation between the LD50 of MLA and the amount of α7 nAChR in various mouse strains, suggesting that mice with more α7 nAChR require more MLA to be poisoned. However, recent research demonstrated that there was no difference in the acute lethality (LD50 ) to MLA in mice lacking the α7 nAChR subunit compared with wild-type mice. The objective of this study was to determine if the α7 nAChR subunit plays a role in motor coordination deficiencies that result from exposure to nAChR antagonists and agonists. We compared the motor function and coordination in wild-type mice to mice lacking the α7 subunit of the nAChR, after treating them with a non-lethal dose of MLA or anabasine, using the following tests: balance beam, grip strength, rotarod, open field and tremor monitor. Analysis of the data indicated that overall there was no difference between the wild-type and knockout mice (P = 0.39 for grip strength; P = 0.21 for rotarod; P = 0.41 for balance beam; P = 0.22 for open field; and P = 0.62 for tremors). Thus results from this study suggest that α7 nAChR does not play an integral role in the acute effects of MLA or anabasine on motor function/coordination. Consequently other subunits of nAChRs found in the neuromuscular junction are likely the primary target for MLA and anabasine resulting in motor coordination deficiencies and acute toxicosis.

Cyclopamine-induced synophthalmia in sheep: defining a critical window and toxicokinetic evaluation.

Cyclopamine, a steroidal alkaloid, from the plant Veratrum californicum is teratogenic, causing a range of different birth defects. The critical window for cyclopamine-induced synophthalmia formation has been reported to be gestational day (GD) 14. The objectives of this study were to better describe cyclopamine-induced craniofacial deformities, to better define the window of susceptibility to synophthalmia formation, and to characterize cyclopamine toxicokinetics in sheep. Ewes were dosed i.v. with purified cyclopamine for toxicokinetic analysis. Another four groups of ewes were dosed orally twice daily with 0.88 g/kg of V. californicum on GD 13, 14 or 15 or consecutively on GD days 13-15. Pregnancy and pre-partum fetal malformations were determined by ultrasound imaging on GD 60. At parturition lambs were assessed for gross malformations. The elimination half-life of cyclopamine in ewes was determined to be 1.1 +/- 0.1 h. The rapid clearance of cyclopamine indicates that ingestion of V. californicum must occur during a very narrow window for synophthalmia formation to occur. Ewes dosed with V. californicum on GD 13 or 14 had lambs with various craniofacial malformations including cyclopia, maxillary dysplasia and mandibular micrognathia. Ewes dosed on GD 15 delivered normal lambs. Ewes dosed consecutively on GD 13-15 were not pregnant at GD 60 and Veratrum-induced embryonic death was assumed to be the cause. Interestingly, lambs with cyclopia were smaller, under-developed and appeared premature even though their twin appeared fully developed. Initial evaluations suggest this was due to placental dysplasia.

Maximising biogas in anaerobic digestion by using engine waste heat for thermal hydrolysis pre-treatment of sludge.

Dublin's Ringsend WWTP was designed to serve a population of approximately 1.2 million p.e. with a sludge production of 37,000 dry tonnes per year after upgrading to full secondary treatment. Several technical solutions were put forward as part of a design, build, finance and operate (DBFO) competition, with the chosen solution being a proposal by Black and Veatch for a combination of sequencing batch reactor (SBR) technology and anaerobic digestion with Cambi thermal hydrolysis pre-treatment (THP). The THP plant was built by Cambi and handed over to B&V in 2002. The plant is now operated by Celtic Anglian Water. In September 2004 a test was carried out on the mass and energy balance of the plant following 2 years of operation and is detailed in this paper. The process enables digestion at very high dry solids feed and low hydraulic retention time. The plant was built with three digesters of 4250 m3 each and is fed with hydrolysed sludge at 11% DS. There are four no. 1 MW Jenbacher engines operating mainly on biogas. Each pair of engines is fitted with a waste heat boiler with a capacity of one tonne steam per hour. These boilers have sufficient capacity to provide 80% of the steam required for the THP, which in turn provides all the heat for the subsequent digestion in the form of hydrolysed feed. There are two main biogas boilers for top up steam and other uses of the biogas including thermal oxidation of concentrated odours.

Effect of protein supplementation on forage utilization by cattle in annual grass-dominated rangelands in the Channeled Scablands of eastern Washington.

Medusahead [ (L.) Nevski] has become a major invasive plant on the annual grass-dominated rangelands within the Channeled Scablands of eastern Washington. Livestock typically avoid grazing medusahead, and forage alternatives are becoming limited in the region. Our hypothesis was that supplying a high-CP supplement would provide a nutritional context that complements the nutritional composition of medusahead and other annual grasses and thus aid cattle in utilizing this vegetation component, making grazing a more effective method of weed control. Cattle grazed annual grass-infested rangelands dominated with medusahead for 10-d periods during June, July, and August over 2 consecutive years. Eight separate pastures were grazed by cattle pairs during each of the 3 grazing periods. Cattle in 4 control pastures received no supplement and cattle in another 4 pastures received a supplement of canola meal that supplied 75% of the daily recommended CP requirement. Bite counts were used to determine diet composition. Forage categories consisted of annual grasses, perennial grasses, and forbs. Bites taken of annual grasses were similar between treatment groups during the first 5 d of the grazing period ( > 0.05), and then cattle supplemented with canola meal increased consumption of annual grasses, during d 6 to 10 of the grazing periods, over nonsupplemented animals ( < 0.05). Consumption of annual grasses was greater during the second year of the grazing study ( < 0.05), likely due to a decline in abundance of forage alternatives in the plant community. The percentage of medusahead in the annual grass forage class tended to decrease in grazed pastures over the 3 yr of the study ( = 0.056): 87 ± 4.2, 64 ± 3.6, and 50 ± 3.6%, respectively. The percentage of medusahead in the annual grasses was similar across years in nongrazed pastures ( > 0.05). Forb production was greatest the first year of grazing and declined the second year of grazing and continued to decline the following year with no grazing ( < 0.05). Perennial grass production was low throughout the study. The effects of grazing on medusahead abundance suggest cattle may be used to graze this weed after it has matured in an integrated management program with other forms of control to reduce infestation prior to seeding with desirable forage species.

Effects of steroidal glycoalkaloids from potatoes (Solanum tuberosum) on in vitro bovine embryo development.

alpha-Solanine and alpha-chaconine are two naturally occurring steroidal glycoalkaloids in potatoes (Solanum tuberosum), and solanidine-N-oxide is a corresponding steroidal aglycone. The objective of this research was to screen potential cyto-toxicity of these potato glycoalkaloids using bovine oocyte maturation, in vitro fertilization techniques and subsequent embryonic development as the in vitro model. A randomized complete block design with four in vitro oocyte maturation (IVM) treatments (Experiment 1) and four in vitro embryo culture (IVC) treatments (Experiment 2) was used. In Experiment 1, bovine oocytes (n=2506) were matured in vitro in medium supplemented with 6 microM of alpha-solanine, alpha-chaconine, solanidine-N-oxide or IVM medium only. The in vitro matured oocytes were then subject to routine IVF and IVC procedures. Results indicated that exposure of bovine oocytes to the steroidal glycoalkaloids during in vitro maturation inhibited subsequent pre-implantation embryo development. Potency of the embryo-toxicity varied between these steroidal glycoalkaloids. In Experiment 2, IVM/IVF derived bovine embryos (n=2370) were cultured in vitro in medium supplemented with 6 microM of alpha-solanine, alpha-chaconine, solanidine-N-oxide or IVC medium only. The results showed that the pre-implantation embryo development is inhibited by exposure to these glycoalkaloids. This effect is significant during the later pre-implantation embryo development period as indicated by fewer numbers of expanded and hatched blastocysts produced in the media containing these alkaloids. Therefore, we conclude that in vitro exposure of oocytes and fertilized ova to the steroidal glycoalkaloids from potatoes inhibits pre-implantation embryo development. Furthermore, we suggest that ingestion of Solanum species containing toxic amounts of glycoalkaloids may have negative effects on pre-implantation embryonic survival.

Elk (Cervus Canadensis) preference for feeds varying in selenium concentration.

Selenium-accumulator plants are reputed to be unpalatable to large ungulates. Elk (Cervus canadensis) populations in southeastern Idaho overlap with populations of Se-rich plants, but there is no information on the influence of plant Se concentration on elk dietary preferences. The objective of this study was to determine, under controlled conditions, the preference of elk for feeds varying in Se concentrations. Seven yearling female elk (128 ± 5 kg) were purchased from a commercial elk farm in southeastern Idaho and adapted to low-Se alfalfa pellets. Three feeding trials using pellets with predetermined Se concentrations were conducted. Alfalfa pellets were commercially made with the addition of Symphyotrichum ascendens (western aster) so that the pellets contained 0.4, 5, 20, 50, or 100 mg/kg (DM basis) Se. In trial 1, 5 Se-containing alfalfa pellets (0.4, 5, 20, 50, and 100 mg/kg Se) were offered for 10 d; trial 2 used 4 Se-containing alfalfa pellet choices (0.4, 20, 50, and 100 mg/kg), and trial 3 used 3 pellet choices (0.4, 50, and 100 mg/kg) for 6 d. In trial 1, consumption of the control pellets by elk was greater than each of the other pellet choices (P < 0.001). Similarly, consumption of the 5-mg/kg Se pellet differed from control pellet and all other Se-containing pellets (P < 0.0001). There were no differences (P > 0.50) in consumption of the 20-, 50-, or 100-mg/kg Se pellets. In trial 2, elk consumed more (P < 0.0001) of the control pellet than the 20-, 50-, and 100-mg/kg Se pellets. Similarly, elk consumed more (P < 0.0001) of the 20-mg/kg Se pellet than the 50- and 100-mg/kg Se pellets. There were no differences (P > 0.99) in elk consumption of the 50- and 100-mg/kg Se pellets. In trial 3, elk consumption of the control and 50- and 100-mg/kg Se pellets differed (P ≤ 0.03) from one another each day except that on d 1 and 2, where elk consumption of the 50- and 100-mg/kg Se pellets did not differ (P ≥ 0.32). Elk clearly discriminated against pellets with the highest Se concentrations when they were given pellets with differing Se concentrations. These results suggest that elk are not likely to select forages with high Se concentrations, particularly when high-Se plants are present in a rangeland situation with numerous other forage choices.

The effect of intermittent dosing of Nicotiana glauca on teratogenesis in goats.

Sustained inhibition of fetal movement in livestock species, induced by several poisonous plants, can result in numerous skeletal-contracture malformations. Lupines are responsible for a condition in cattle referred to as "crooked calf syndrome" that occurs when pregnant cattle graze teratogenic lupines. Similar malformations are also seen in animals poisoned by Conium maculatum (coniine) and Nicotiana glauca (anabasine). A proposed management strategy to limit these types of birth defects includes utilizing an intermittent grazing schedule to allow short durations of grazing lupine-infested areas interrupted by movement to a lupine-free pasture. The objective of this study was to use a goat model to determine if an intermittent schedule of five continuous days on treatment followed by two days off treatment would be sufficient to decrease, or prevent, the incidence of anabasine-induced malformations. The data from this study suggest that, for N. glauca in goats, the intermittent grazing program of five days exposure with two days of non-exposure is insufficient to prevent significant skeletal malformations from occurring. However, this study did demonstrate an inverse relationship between the amount of serum anabasine in the dam and the extent of fetal movement.

Congenital skeletal malformations and cleft palate induced in goats by ingestion of Lupinus, Conium and Nicotiana species.

Three piperidine alkaloid containing plants, Conium maculatum (poison-hemlock), Nicotiana glauca (tree tobacco) and Lupinus formosus (lunara lupine), induced multiple congenital contractures (MCC) and palatoschisis in goat kids when their dams were gavaged with the plant during gestation days 30-60. The skeletal abnormalities included fixed extension or flexure of the carpal, tarsal, and fetlock joints, scoliosis, lordosis, torticollis and rib cage abnormalities. Clinical signs of toxicity included those reported in sheep, cattle and pigs--ataxia, incoordination, muscular weakness, prostration and death. One quinolizidine alkaloid containing plant, Lupinus caudatus (tailcup lupine), on the other hand, which is also known to cause MCC in cows, caused only slight signs of toxicity in pregnant goats and no teratogenic effects in their offspring.

Garlic in the ovine fetal environment.

We investigated whether the odor of garlic can cross the placental barrier from the mother to the fetal environment. Samples of amniotic fluid, allantoic fluid, fetal blood, and maternal blood were collected 0, 50, 100, and 150 min after a pregnant ewe (approximately day 110 of gestation) was gavaged with 6 ml of Egyptian garlic oil. A panel of judges detected (p < 0.05) garlic odor in samples of allantoic fluid, fetal blood, and maternal blood collected 50, 100, and 150 min after the ewe was given garlic and in samples of amniotic fluid collected 100 min after treatment.

Concentration of galegine in Verbesina encelioides and Galega oficinalis and the toxic and pathologic effects induced by the plants.

Verbesina encelioides administered to sheep by gavage induced clinical signs of toxicity and pathologic lesions identical to those induced by Galega officinalis. Sheep had compromised respiratory function with shallow, rapid respiration and frothy exudate from the nares. Affected animals necropsied at time of death presented with hydrothorax with as much as 2 to 3 L of straw-colored thoracic fluid with fibrin tags and congestion and edema of the lungs. The trachea and lung airways contained frothy material with fibrin strands. In some cases, subendocardial hemorrhage of the left ventricle was present. Galegine, a guanidine compound believed to be responsible for these effects, was found at an average concentration of about 0.46% in Galega and at 0.08% in the Verbesina collection that induced toxicosis. While G. officinalis is a known poisonous plant, its very limited distribution in the U.S. causes it to be of minor importance. V. encelioides, however, is widely distributed in the U.S. and presents a potential hazard for grazing livestock. Verbesina may have been responsible for past livestock deaths in the U.S., and thus should be classified as a poisonous plant.

Pine needle abortion in cattle: metabolism of isocupressic acid.

The rumen and hepatic metabolism of the cattle abortifacient compound isocupressic acid (ICA) was examined in vitro and in vivo. ICA was incubated for 56 h in bovine rumen inoculum and was found to be converted to three compounds identified as imbricatoloic acid, a structurally uncharacterized isomer of imbricatoloic acid, and dihydroagathic acid. In preparations of liver homogenates, ICA was found to be oxidized to agathic acid. No differences in ICA metabolites were detected in comparing the cow, sheep, pig, goat, guinea pig, and rat livers; however, guinea pig and rat liver homogenates were less efficient in converting ICA to agathic acid. ICA had been administered to cows orally and by intravenous infusion and induced abortions after either method of treatment. After intravenous infusion, agathic acid was identified as the major metabolite together with minor amounts of dihydroagathic acid. After oral administration, dihydroagathic acid was identified as the major metabolite with minor amounts of agathic acid, imbricatoloic acid, and a structurally uncharacterized metabolite tentatively identified as tetrahydroagathic acid.

Analysis of toxic norditerpenoid alkaloids in Delphinium species by electrospray, atmospheric pressure chemical ionization, and sequential tandem mass spectrometry.

A rapid electrospray mass spectrometry method was developed for screening larkspur (Delphinium spp.) plant material for toxic norditerpenoid alkaloids. The method was calibrated using two standard alkaloids, methyllycaconitine (1) and deltaline (2), with a recovery of 92% from spiked samples and relative standard deviations of 6.0% and 8.1% for the two alkaloids, respectively. Thirty-three samples of plains larkspur, Delphinium geyeri, were analyzed. Methyllycaconitine (1) concentration was 0.27% +/- 0.08% during a 1-month period in 1997 establishing the relative risk of poisoning from the plant to be low. The method was also applied to the trace analysis (<1 ppm) of 1 in serum samples from sheep dosed different levels of the alkaloid. Electrospray ionization combined with sequential tandem mass spectrometry and HPLC coupled to atmospheric pressure chemical ionization (APCI) mass spectrometry were used to detect and tentatively identify three new norditerpenoid alkaloids from Delphinium nuttallianum [bearline (6), 14-acetylbearline (7), 16-deacetylgeyerline (8)]. The tentative structure of the new alkaloids was predicted from the tandem mass spectra fragmentation patterns and assigning the substitution pattern for methoxy and acetyl groups at the C-14 and C-16 carbons.

Water hemlock (Cicuta douglasii) toxicoses in sheep: pathologic description and prevention of lesions and death.

Water hemlock causes numerous livestock losses in North America every year. Description of pathologic and serum biochemical changes has been lacking in the literature. Tubers of western water hemlock (Cicuta douglasii) induced excessive salivation, tremors, grand mal seizures, skeletal and cardiac myodegeneration, and death in sheep given 1.2-2.7 g fresh tuber/kg body weight by gavage. Seizures were intermittent with periods of relaxation until death occurred from anoxia during seizure activity. In sheep given 1.5-2.5 times the lethal dose of water hemlock by gavage, intravenous administration of sodium pentobarbital at the onset of the first seizure prevented further seizure activity and skeletal and cardiac myodegeneration and resulted in rapid and complete recovery.

Dose response of sheep poisoned with locoweed (Oxytropis sericea).

Locoweed poisoning occurs when livestock consume swainsonine-containing Astragalus and Oxytropis species over several weeks. Although the clinical and histologic changes of poisoning have been described, the dose or duration of swainsonine ingestion that results in significant or irreversible damage is not known. The purpose of this research was to document the swainsonine doses that produce clinical intoxication and histologic lesions. Twenty-one mixed-breed wethers were dosed by gavage with ground Oxytropis sericea to obtain swainsonine doses of 0.0, 0.05, 0.1, 0.2, 0.4, 0.8, and 1.0 mg/kg/day for 30 days. Sheep receiving > or = 0.2 mg/kg gained less weight than controls. After 16 days, animals receiving > or = 0.4 mg/kg were depressed, reluctant to move, and did not eat their feed rations. All treatment groups had serum biochemical changes, including depressed alpha-mannosidase, increased aspartate aminotransferase and alkaline phosphatase, as well as sporadic changes in lactate dehydrogenase, sodium, chloride, magnesium, albumin, and osmolarity. Typical locoweed-induced cellular vacuolation was seen in the following tissues and swainsonine doses: exocrine pancreas at > or = 0.05 mg/kg; proximal convoluted renal and thyroid follicular epithelium at > or = 0.1 mg/kg; Purkinje's cells, Kupffer's cells, splenic and lymph node macrophages, and transitional epithelium of the urinary bladder at > or = 0.2 mg/kg; neurons of the basal ganglia, mesencephalon, and metencephalon at > or = 0.4 mg/kg; and cerebellar neurons and glia at > or = 0.8 mg/kg. Histologic lesions were generally found when tissue swainsonine concentrations were approximately 150 ng/g. Both the clinical and histologic lesions, especially cerebellar lesions are suggestive of neurologic dysfunction even at low daily swainsonine doses of 0.2 mg/kg, suggesting that prolonged locoweed exposure, even at low doses, results in significant production losses as well as histologic and functional damage.

The fetal cleft palate: II. Scarless healing after in utero repair of a congenital model.

The role of fetal surgery in the treatment of non-life-threatening congenital anomalies remains a source of much debate. Before such undertakings can be justified, models must be established that closely resemble the respective human anomalies, and the feasibility and safety of these in utero procedures must be demonstrated. The authors recently described and characterized a congenital model of cleft palate in the goat. The present work demonstrates the methodology they developed to successfully repair these congenital cleft palates in utero, and it shows palatal healing and development after repair. A surgically created cleft model was developed for comparative purposes. Palatal shelf closure normally occurs at approximately day 38 of gestation in the caprine species. Six pregnant goats were gavaged twice daily during gestational days 32 to 41 (term, 145 days) with a plant slurry of Nicotiana glauca containing the piperidine alkaloid anabasine; the 12 fetuses had complete congenital clefts of the secondary palate. Repair of the congenital clefts was performed at 85 days of gestation using a modified von Langenbeck technique employing lateral relaxing incisions with elevation and midline approximation of full-thickness, bilateral, mucoperiosteal palatal flaps followed by single-layer closure. Six congenitally clefted fetuses underwent in utero repair, six remained as unrepaired controls. Twelve normal fetuses underwent surgical cleft creation by excision of a 20 x 3 mm full-thickness midline section of the secondary palate extending from the alveolus to the uvula, at 85 days of gestation. Six surgically clefted fetuses underwent concurrent repair of the cleft at that time; six clefted fetuses remained as unrepaired controls. At 2 weeks of age, no congenitally or surgically created clefts repaired in utero demonstrated gross or histologic evidence of scar formation. A slight indentation at the site of repair was the only remaining evidence of a cleft. At 6 months of age, normal palatal architecture, including that of mucosal, muscular, and glandular elements, was seen grossly and histologically. Cross-section through the mid-portion of the repaired congenitally clefted palates demonstrated reconstitution of a bilaminar palate, with distinct oral and nasal mucosal layers, after single-layer repair. In utero cleft palate repair is technically feasible and results in scarless healing of the mucoperiosteum and velum. The present work represents the first in utero repair of a congenital cleft palate model in any species. The use of a congenital cleft palate model that can be consistently reproduced with high predictability and little variation represents the ideal experimental situation. It provides an opportunity to manipulate specific variables, assess the influence of each change on the outcome and, subsequently, extrapolate such findings to the clinical arena with a greater degree of relevance.

The fetal cleft palate: I. Characterization of a congenital model.

Any animal model of a human congenital anomaly established by iatrogenic methods involving intrauterine fetal manipulation has limited clinical applicability. A congenital model that more closely simulates the etiopathogenesis of a human anomaly may provide data that can more readily be extrapolated to that anomaly and, therefore, be used in diagnostic and management strategies. The present work provides a description and characterization of a congenital model of cleft palate in the goat. Palatal shelf closure normally occurs at approximately day 38 of gestation in the caprine species. Sixteen pregnant goats were gavaged twice daily during gestational days 32 through 41 [term, 145 days] with a plant slurry of Nicotiana glauca containing the piperidine alkaloid teratogen anabasine. Gross analysis and measurement of fetal clefts were performed at 60, 70, and 85 days gestation (four fetuses were studied at each time point). Seventeen clefted kids were sacrificed at specific intervals after birth (2 weeks, and 1, 3, and 6 months); after skull debridement and preparation, they were compared with 12 unclefted control kids. Complete clefting of the secondary palate occurred in 97 percent of the fetuses. In all cases, the cleft extended from the posterior aspect of the alveolar ridge to the uvula; the majority of these clefts were bilateral, with complete detachment of the vomer. Morphologically, these clefts were similar to human clefts. Eighteen percent of clefted newborn kids demonstrated gross maxillary hypoplasia and midfacial retrusion at birth with a relative Class III malocclusion. Direct measurement of the congenital caprine skulls confirmed these findings. The incidence of midfacial growth abnormalities in these clefted animals raises questions regarding the etiopathogenesis of facial dysmorphology that is unrelated to scarring of the maxilla. This congenital cleft palate model is currently being used to explore these questions and others related to craniofacial growth and palatal function after in utero repair.

The effects of pokeweed mitogen (PWM) and phytohemagglutinin (PHA) on bovine oocyte maturation and embryo development in vitro.

The effects of two commonly used cell culture mitogens, pokeweed (PWM) and phytohemagglutinin (PHA) on bovine oocyte maturation in vitro (IVM) and preimplantation embryo development in vitro were evaluated by randomized complete block experimental design with three treatments. Effects were measured by quantifying subsequent embryo development. Oocyte maturation was adversely affected by PWM-containing medium as indicated by a decrease in cleavage rate and subsequent embryo development to morula and blastocyst stages. Embryo developmental competence was also adversely affected by PWM. Development in PHA-containing medium was significantly better (P<0.05) than in the PWM treatment, although there was no difference (P>0.05) when compared to Control. We conclude that there are no beneficial effects in adding mitogenic agents to culture medium to enhance in vitro embryo production and development.

Efects of the pine needle abortifacient, isocupressic acid, on bovine oocyte maturation and preimplantation embryo development.

Isocupressic acid (ICA) [15-hydroxylabda-8 (17), 13E-dien-19-oic acid], a labdane diterpene acid, isolated from ponderosa pine (Pinus ponderosa), Lodgepole pine (Pinus contorta), common juniper (Juniperus communis) and Monterey cypress (Cupressus macrocarpa), induces abortion in pregnant cows when ingested primarily during the last trimester. The objective of this study was to investigate the effects of isocupressic acid on bovine oocyte maturation (in vitro maturation (IVM)-Experiment I) and preimplantation embryo development (in vitro culture (IVC)-Experiment II) using in vitro embryo production techniques and to subsequently evaluate viability and developmental competence of ICA-cultured embryos via embryo transfer to recipient heifers (Experiment III). A complete randomized block experimental design was used. In Experiment I and II, isocupressic acid was added to IVM or IVC media at 0 (TRT1, control), 1.3 (TRT2), and 2.6 microg/ml (TRT3) Results from Experiment I and II indicated that ICA did not inhibit oocyte maturation and did not adversely affect preinpiantation embryo development. Furthermore, results from Experiment II demonstrated that isocupressic acid enhanced bovine preimplantation embryo development in vitro in a dose dependent manner. Subsequently, Day 8 (Day 0 = IVF) blastocysts cultured in vitro in the medium containing 2.6 microg/ml ICA were transferred to recipient heifers and resulted in normal pregnancies as determined by ultrasound imaging. Subsequently, all but two births were normal as evaluated by post natal veterinary examination. In conclusion, ICA showed no adverse effects on oocyte maturation and preimplantation embryo development in vitro or subsequent viability in vivo using the ICA concentrations and in vitro culture parameters of this study.

Development and viability of bovine preplacentation embryos treated with swainsonine in vitro.

This study investigated the effects of swainsonine (a locoweed toxin) on bovine preplacentation embryo development using in vitro procedures. We examined and confirmed the viability and developmental potential of swainsonine-treated embryos by transfer to synchronized recipient heifers. Oocytes (n = 6338) were aspirated from ovaries collected from the abattoir and subjected to in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC). Swainsonine was added to IVM, IVF, IVC media spatially and IVM/IVF/IVC continuously, at 0 ng/ml (TRTI, control), 200 ng/ml (TRT2), 400 ng/ml (TRT3), and 800 ng/ml (TRT4). Embryo development was evaluated with respect to oocyte cleavage rate and the rates of morula and blastocyst formation. There was no difference (P > 0.05) among treatments. The average number of nuclei per blastocyst at Day 7.5 of culture (Day 0 = IVF) was 85.9 +/- 4.3 (n = 47) and 89.3 +/- 4.4 (n = 44) for swainsonine-treated embryos (800 ng/ml) and control embryos, respectively. Pregnancy rate as determined by ultrasonography on day 35 to 40 post embryo transfer was 43.8% and 38.3% for swainsonine-treated (800 ng/ml) and control embryos, respectively. Nine (9.4%) healthy calves were delivered from heifers receiving swainsonine-exposed and nine (9.6%) from control embryos. No difference (P > 0.05) was detected in number of calves developing from TRT and control embryos. We conclude that swainsonine does not have an adverse effect on the development and viability of preplacentation bovine embryos.