Effect of epidermal growth factor and insulin-like growth factor I on porcine preantral follicular growth, antrum formation, and stimulation of granulosal cell proliferation and suppression of apoptosis in vitro

The object of this study was to investigate the role of epidermal growth factor (EGF) and IGF-I in the regulation of preantral follicular growth, antrum formation, and granulosal cell proliferation/ apoptosis. Porcine preantral follicles were manually dissected and cultured for up to 8 d in Waymouth's (Exp. 1) or alpha-minimum Eagle's essential medium (Exp. 2 and 3) supplemented with 10 microg/mL of transferrin, 100 microg/mL of L-ascorbic acid, and 2 mU/mL of ovine FSH, in the presence (Exp. 1 and 3) or absence (Exp. 2) of 7.5% fetal calf serum. According to the experimental protocol, IGF-I (0, 1, 10, or 100 ng/mL; Exp. 1), or IGF-I (50 ng/mL), EGF (10 ng/mL) and EGF+IGF-I (Exp. 2 and 3) were added to the culture media. In Exp. 1, follicles exhibited a concentration-dependent response (P < 0.05) to IGF-I, with the highest rates of granulosal cell proliferation, follicular integrity, and recovery rate of cumulus cell-oocyte complexes and lowest incidence of apoptosis occurring at the highest IGF-I dose. In Exp. 2 serum-free medium, granulosal cell proliferation was low (1 to 5%), irrespective of whether EGF and/or IGF-I were present and cellular apoptosis was increased (P < 0.05) on d 4 and 8 in the EGF+IGF-I group compared with the addition of either factor alone. In Exp. 3, granulosal cell proliferation was high in all follicles cultured in serum-containing medium for the first 3 d, but fell sharply (P < 0.05) on d 4, except in media containing IGF-I. Collectively, EGF and IGF-I increased granulosal cell proliferation, decreased apoptosis, and promoted follicular antrum formation. These results may provide useful information for developing a preantral follicular culture system in which the oocytes are capable of fertilization and embryonic development.     

Cell Biology Symposium: Zinc finger nucleases to create custom-designed modifications in the swine (Sus scrofa) genome

Engineered zinc finger nucleases (ZFN) are rapidly gaining popularity as a means to enhance the rate and specificity of DNA modifications in plant and animal cells. Repair-mediated gene modification by ZFN is driven by introducing DNA double-strand breaks via a nonspecific nuclease domain linked to a sequence-specific zinc finger nucleotide recognition domain. This review examines the use of ZFN to produce genetically modified swine and the potential of this technology for the future. By combining conventional gene targeting methods with somatic cell nuclear transfer, several genetically modified pig models have been produced. These conventional techniques are inefficient in mammalian somatic cells and provide little control over the site specificity and rate of exogenous DNA integration. The use of engineered ZFN that bind and cleave genomic DNA at specific loci can enhance targeting efficiencies by orders of magnitude. Recent publication of the first genetic modification in pigs by combining ZFN technology with somatic cell nuclear transfer has opened the door to genome targeting with a precision that was not previously possible in a large animal model. Since that time, model pigs with selective knockout of endogenous genes have been produced. This review will examine the use of ZFN to generate these pig models and the potential of ZFN to accelerate the production of genetically modified pigs of agricultural and biomedical importance. Current methods of ZFN design, important considerations for their safe and effective use in modification of the swine genome, and future innovative applications of this technology in pigs will be discussed.     

Oxygen Concentration and Cysteamine Supplementation During In vitro Production of Buffalo (Bubalus bubalis) Embryos Affect mRNA Expression of BCL‐2, BCL‐XL,MCL‐1, BAX and BID

This study examined the effects of O₂ concentration (5% vs 20%) during in vitro maturation (IVM), fertilization (IVF) and culture (IVC) or supplementation of IVM and IVC media with cysteamine (50 and 100 μm , respectively; IVM, IVF and IVC carried out in 20% O₂), on blastocyst rate and relative mRNA abundance of some apoptosis‐related genes measured by real‐time qPCR in immature and in vitro‐matured buffalo oocytes and in embryos at 2‐, 4‐, 8‐ to 16‐cell, morula and blastocyst stages. The blastocyst rate was significantly higher (p < 0.05) while the percentage of TUNEL‐positive cells was significantly lower (p < 0.05) under 5% O₂ than that under 20% O₂. The mRNA expression of anti‐apoptotic genes BCL‐2 and MCL‐1 was significantly higher (p < 0.05) and that of pro‐apoptotic genes BAX and BID was lower (p < 0.05) under 5% O₂ than that under 20% O₂ concentration at many embryonic stages. Following cysteamine supplementation, the blastocyst rate and the relative mRNA abundance of BCL‐XL and MCL‐1 was significantly higher (p < 0.05) and that of BAX but not BID was lower (p < 0.05) at many stages of embryonic development, although it did not affect the percentage of TUNEL positive cells in the blastocysts significantly. The mRNA expression pattern of these genes during embryonic development was different in 5% vs 20% O₂ groups and in cysteamine supplemented vs controls. At the 8‐ to 16‐cell stage, where developmental block occurs in buffalo, the relative mRNA abundance of BCL‐2 and MCL‐1 was highest under 5% O₂ concentration and that of BAX and BID was highest (p < 0.05) under 20% O₂ concentration. These results suggest that one of the mechanisms through which beneficial effects of low O₂ concentration and cysteamine supplementation are mediated during in vitro embryo production is through an increase in the expression of anti‐apoptotic and a decrease in the expression of pro‐apoptotic genes.     

Analysis of the risk of introduction and spread of porcine reproductive and respiratory syndrome virus through importation of raw pigmeat into New Zealand

AIMS: To determine the frequency with which porcine reproductive and respiratory syndrome (PRRS) virus (PRRSv) would become established in a non-commercial pig herd in New Zealand due to illegal feeding of uncooked food waste containing virus-contaminated pigmeat. To determine the likelihood of a single incursion resulting in a multi-farm outbreak of the disease, and describe the spatio-temporal characteristics of such an outbreak.

METHODS: A Monte Carlo simulation model was constructed to determine the expected annual frequency of PRRSv infection being initiated in a non-commercial pig herd as a result of inadvertent feeding of pigmeat imported from countries endemically infected with the disease. Once the likelihood of PRRSv becoming established in a single pig herd was determined, stochastic spatially explicit infectious disease modelling software was utilised to model the temporal and spatial characteristics of the resulting epidemic.

RESULTS: Assuming the proportion of imported pigmeat remained at current levels, consumption patterns of pigmeat in households in New Zealand remained steady, and limited compliance with recently reintroduced regulations to prevent feeding of uncooked food waste, at least 4.3 pig herds per year were predicted to become infected with PRRSv. Simulation modelling of PRRSv epidemics related to initial infection of a non-commercial farm produced an estimate that 36% of these incursions would spread from the initial herd, and that these outbreaks would involve 93 herds on average in the first year. By increasing the estimated persistence of PRRSv infection in small herds, an average of 205 herds became infected in the first year.

CONCLUSIONS: Given a mean of 4.3 infected premises per year and a 36% probability of infection spreading beyond the initial infected herd, there was a 95% likelihood of a multi-farm PRRS outbreak occurring within 3 years.

CLINICAL RELEVANCE: Introduction of PRRSv through importation of virus-contaminated pigmeat presents a high risk for establishment of the disease in the pig industry in New Zealand.     

Effects of intensification of pastoral farming on greenhouse gas emissions in New Zealand

Abstract

AIMS: The aims of this cross-sectional study were to investigate the herd and cow-level prevalence of bovine digital dermatitis (BDD) in dairy farms in the northern Taranaki region of New Zealand, and to identify whether there was any spatial clustering of herds with the disease.

METHODS: A survey of 224 dairy farms in the northern Taranaki region of New Zealand was undertaken from September 2014 to February 2015. Following training in robust criteria to confirm BDD visually, a technician inspected the rear feet of every milking cow on the farms during milking. The identity of cows with lesions and the feet involved were recorded. The proportion of cows affected among the inspected population (cow-level prevalence), the proportion of a herd affected (farm-level prevalence), and proportion of farms with ≥1 cow with lesions, were calculated. A bivariate K function analysis was then used to assess whether farms with ≥1 cow with lesions were clustered, after accounting for the distribution of the farms involved in the study.

RESULTS: Bovine digital dermatitis lesions were observed on 143/224 (63.8 (95% CI=57.5–70.1)%) farms. Within-farm prevalence was 0% on 81 (36.2%) farms, between >0 and <3% on 120 (53.5%) farms, with a maximum prevalence of 12.7% on one farm. Overall, cow-level prevalence was 707/60,455 (1.2 (95% CI=0.9–3.0)%), and on affected farms was 707/41,116 (1.7 (95% CI=1.4–2.1)%). In affected cows, 268/707 (37.9%) had a lesion on left foot only, 262/707 (37.1%) on the right foot only and 177/707 (25.0%) on both feet. The K function analysis showed no evidence of clustering of farms with BDD.

CONCLUSIONS: Bovine digital dermatitis was widespread among the survey farms, but there was no evidence that there was any clustering of herds with BDD. The cow-level prevalence on affected farms was much lower than reported elsewhere.

CLINICAL RELEVANCE: Although the prevalence at the cow level was low, if these data are representative of other regions of New Zealand, BDD could easily become a major problem on dairy farms in New Zealand, as has been observed in other countries.     

Shape transition of germanium nanocrystals on a silicon (001) surface from pyramids to domes

Chemical vapor deposition of germanium onto the silicon (001) surface at atmospheric pressure and 600 degrees Celsius has previously been shown to produce distinct families of smaller (up to 6 nanometers high) and larger (all approximately 15 nanometers high) nanocrystals. Under ultrahigh-vacuum conditions, physical vapor deposition at approximately the same substrate temperature and growth rate produced a similar bimodal size distribution. In situ scanning tunneling microscopy revealed that the smaller square-based pyramids transform abruptly during growth to significantly larger multifaceted domes, and that few structures with intermediate size and shape remain. Both nanocrystal shapes have size-dependent energy minima that result from the interplay between strain relaxation at the facets and stress concentration at the edges. A thermodynamic model similar to a phase transition accounts for this abrupt morphology change.     

A kuroko-type polymetallic sulfide deposit in a submarine silicic caldera

Manned submersible studies have delineated a large and actively growing Kuroko-type volcanogenic massive sulfide deposit 400 kilometers south of Tokyo in Myojin Knoll submarine caldera. The sulfide body is located on the caldera floor at a depth of 1210 to 1360 meters, has an area of 400 by 400 by 30 meters, and is notably rich in gold and silver. The discovery of a large Kuroko-type polymetallic sulfide deposit in this arc-front caldera raises the possibility that the numerous unexplored submarine silicic calderas elsewhere might have similar deposits.