Katherine Hendricks

Effects of gamete source and culture conditions on the competence of in vitro-produced embryos for post-transfer survival in cattle

One limitation to the use of in vitro-produced embryos in cattle production systems is the fact that pregnancy rates after transfer to recipients are typically lower than when embryos produced in vivo are transferred. Conceptually, the oocyte and spermatozoon from which the embryo is derived could affect competence for post-transfer survival. There are sire differences in embryonic survival after transfer, but there is little evidence that an embryos ability to establish pregnancy is determined by sex sorting of spermatozoa by flow cytometry. The role of the source of the oocyte as a determinant of embryonic survival after transfer has not been examined carefully. Conditions for embryo culture after fertilisation can have an impact on the ability of the embryo to establish pregnancy following transfer. Among the specific molecules produced in the reproductive tract of the cow that have been shown to improve competence of in vitro-produced embryos for post-transfer survival are colony-stimulating factor 2, insulin-like growth factor-1 (for recipients exposed to heat stress) and hyaluronan (for less-advanced embryos). There is also a report that embryo competence for post-transfer survival can be improved by inclusion of a carbon-activated air filtration system in the incubator used to culture embryos. Progress in developing culture systems to improve embryonic competence for survival after transfer would be hastened by the development of in vitro assays that accurately predict the potential of an embryo to establish pregnancy after transfer. A group of 52 genes has been identified that are differentially expressed in embryos that developed to term v. embryos that did not establish pregnancy. Perhaps a gene microarray consisting of these genes, alone or in combination with other genes, could be used to screen embryos for competence to establish pregnancy.

New report of Colletotrichum gloeosporioides causing postbloom fruit drop on citrus in Bermuda

Abstract Postbloom fruit drop (PFD) of citrus was observed for the first time after a widespread and severe outbreak occurred in Bermuda in the 1990s. Fruit losses from the disease were estimated at 25% to 35% in sweet orange, grapefruit, lemon and Tahiti lime. The causal agent of PFD has been reported as either Colletotrichum gloeosporioides or Colletotrichum acutatum. Bermuda isolates of Colletotrichum recovered from diseased orange trees produced lesions typical of PFD in detached petals of orange, lime and grapefruit, and in attached orange blossoms. The isolates produced few to no lesions of anthracnose on leaves of key lime seedlings. Kochs postulates were fulfilled following reisolation of morphologically identical fungi from inoculated tissues. The isolates were characterized using morphological (conidial size and shape and colony colour), physiological (growth rate at 24 °C and on a benomyl-amended medium), immunological (ELISA) and molecular (PCR amplification and sequencing of the ITS region) methods. Immunological and molecular techniques provided definitive identification of the isolates as Colletotrichum gloeosporioides. This report confirms that C. gloeosporioides can be the causal agent of PFD; therefore, identification of the causal agent to species by immunology, molecular analysis and fungicide sensitivity is suggested for new outbreaks of the disease.

Can programmed cell death be induced in post-ejaculatory bull and stallion spermatozoa?

Apoptosis is common during spermatogenesis. Here, it was tested whether apoptosis could be induced in sperm after ejaculation. There were several lines of evidence to indicate that sperm are resistant to induction of apoptosis. First, incubation of bull sperm at temperatures characteristic of normothermia (38.5 degrees C) or heat shock (40 and 41 degrees C) for 4h did not increase the proportion of sperm positive for the TUNEL reaction. There was also no reduction in mitochondrial polarity caused by exposure to 40 or 41 degrees C. Incubation at 38.5 degrees C (least-squares mean+/-SEM=4.0+/-1.4%), 40 degrees C (6.2+/-1.4%), and 41 degrees C (7.0+/-1.4%) for 24h did increase the proportion of sperm that were TUNEL positive slightly as compared to non-incubated control sperm (1.0+/-1.4%). However, the increase in TUNEL labeling was not affected by incubation temperature and occurred even in the presence of the group II caspase inhibitor, z-DEVD-fmk. In addition, exposure of bull sperm to carbonyl cyanide 3-chlorophenylhydrazone (CCCP), which depolarizes mitochondrial membranes, did not increase TUNEL labeling. Stallion sperm were also resistant to increased TUNEL labeling in response to incubation at 41 degrees C for 4h or exposure to CCCP. Western blotting was performed to determine whether failure of induction of apoptosis was due to aberrant caspase activation. Procaspase-9 was detected in bull sperm, but cleavage to caspase-9 was not induced by short-term aging at 38.5, 40, or 41 degrees C, or exposure to CCCP. Procaspase-3 was not detected in bull spermatozoa. In conclusion, post-ejaculatory bull and stallion sperm were resistant to induction of apoptosis; this resistance, at least in bulls, was due to refractoriness of mitochondria to heat shock-induced depolarization, lack of activation of procaspase-9, and an absence of procaspase-3.

The block to apoptosis in bovine two-cell embryos involves inhibition of caspase-9 activation and caspase-mediated DNA damage

The capacity of the preimplantation embryo to undergo apoptosis in response to external stimuli is developmentally regulated. Acquisition of apoptosis does not occur in the cow embryo until between the 8- and 16-cell stages. The purpose of the present experiments was to determine the mechanism by which apoptosis is blocked in the bovine two-cell embryo. Heat shock (41 degrees C for 15 h) did not increase activity of caspase-9 or group II caspases (caspase-2, -3, and -7) in two-cell embryos but did in day 5 embryos. Exposure of embryos to carbonyl cyanide 3-chlorophenylhydrazone (CCCP) to depolarize mitochondria resulted in activation of caspase-9 and group II caspases at both stages of development. For day 5 embryos, CCCP also increased the proportion of blastomeres that underwent DNA fragmentation as determined by the TUNEL assay. In contrast, CCCP did not increase TUNEL labeling when applied at the two-cell stage. In conclusion, failure of heat shock to increase caspase-9 and group II caspase activity in the two-cell embryo indicates that the signaling pathway leading to mitochondrial depolarization and caspase activation is inhibited at this stage of development. The fact that CCCP treatment of two-cell embryos induced caspase-9 and group II-caspase activity indicates that caspase activation is possible following mitochondrial depolarization. However, since CCCP did not increase TUNEL labeling of two-cell embryos, actions of group II-caspases to activate DNases is inhibited.

Host specificity testing and examination for plant pathogens reveals that the gall-inducing psyllid Calophya latiforceps is safe to release for biological control of Brazilian peppertree

Brazilian peppertree, Schinus terebinthifolia Raddi (Anacardiaceae), is one of the worst upland exotic weeds in Florida, USA. Foreign exploration for natural enemies led to the discovery of a pit‐galling psyllid, Calophya latiforceps Burckhardt (Hemiptera: Calophyidae), in the state of Bahia, Brazil, in 2010. Crawlers of C. latiforceps stimulate the formation of galls on the leaves of S. terebinthifolia resulting in leaf discoloration and in some cases leaf abscission. To determine whether C. latiforceps is a safe candidate for biological control of S. terebinthifolia, host specificity and the presence of selected plant pathogens were examined. Adult oviposition, gall formation, and adult survival of C. latiforceps were examined on 89 plant species under no‐choice and choice conditions. We found that C. latiforceps laid eggs on plants in seven families; however, crawlers stimulated gall formation and completed development to adult only on S. terebinthifolia. All crawlers on non‐target plants died, likely due to starvation caused either by the absence of a feeding stimulus or by a hypersensitive plant response. Under no‐choice conditions, 10% of adults lived for 19 days on the target weed, but adult survival was reduced to <3 days on non‐target plants. Choice testing revealed that females preferred to oviposit on S. terebinthifolia compared to non‐target plants. Molecular methods and indicator host inoculations did not detect the presence of ‘Candidatus Liberibacter solanacearum’, ‘Ca. L. asiaticus’, ‘Ca. L. americanus’, ‘Ca. L. africanus’, or plant viruses in adult C. latiforceps. We conclude that releasing C. latiforceps in the USA will have extremely low risk to non‐target plants, and provides another tool for the management of S. terebinthifolia.

Effects of airport screening X-irradiation on bovine sperm chromatin integrity and embryo development

Biological samples, including cryopreserved sperm, are routinely X-rayed during air shipment. The goal was to investigate the impact of X-irradiation used for checked and carry-on luggage on bovine sperm chromatin integrity and postfertilization in vitro embryonic development. Frozen domestic bull sperm (Bos taurus) (n=9 bulls) stored in a dry shipper (-160 degrees C) was screened by X-irradiation 0, 1, 2, and 3 times as either carry-on or checked luggage. Duplicate straws were thawed, and sperm were assessed for chromatin damage using the sperm chromatin structure assay (SCSA) and by postfertilization in vitro developmental competence of mature oocytes. Multiple exposure to X-rays did not significantly affect sperm chromatin integrity assessed by SCSA. There were lower proportions of oocytes cleaved (P=0.07; 21.6+/-3.1% vs. 29.4+/-3.1%, 24.9+/-3.1%, and 25.7+/-3.3% for 3 vs. 0, 1, and 2 times, respectively; least-squares means+/-SEM) and that developed to blastocysts (P=0.06; 9.0+/-1.7% vs. 13.8+/-1.7%, 11.5+/-1.7%, and 12.6+/-1.9%, respectively) when fertilization was performed with sperm X-rayed 3 times using checked luggage irradiation; developmental competence (percentage cleaved embryos becoming blastocysts) was unaffected. There were no deleterious effects of other X-irradiation treatments on embryo development. We inferred that screening by X-irradiation may reduce the ability of sperm to activate oocyte cleavage after multiple exposures at the checked luggage dose. However, there was no evidence that competence of embryos to become blastocysts was reduced by X-irradiation (45.4+/-5.7%, 40.4+/-5.7%, 46.4+/-6.1%, and 41.8+/-5.7% for 0, 1, 2, and 3 doses, respectively), but potential long-term epigenetic effects are unknown.

Spatial and Temporal Patterns of Commercial Citrus Trees Affected by Phyllosticta citricarpa in Florida

Citrus black spot (CBS) caused by Phyllosticta citricarpa, is the most recent introduction of an exotic citrus pathogen into Florida and has been a challenge to control to date. Understanding the dispersal pattern of the disease within affected groves is vital in developing effective control strategies to limit the spread of the disease. The spatial pattern of CBS-affected trees was studied in two commercial ‘Valencia’ orange groves over three consecutive citrus seasons. Cluster analyses based on nearest-neighbor distance (F, G and J-functions) and pairwise distances between points (Ripley’s K function, Besag’s L function and the pair correlation function, g) were used to test the hypothesis of complete spatial randomness (CSR) of CBS infected trees within the groves. In both groves, the hypothesis of CSR was rejected for all tests performed including quadrats testing (2 × 2 trees up to 10 × 10 trees). The relationship between tree age and disease was assessed at one experimental site. Citrus trees bearing fruit for the first time accounted for approximately 13% of trees positive for disease and were located within areas of heavy disease pressure. These findings support short distance movement of inoculum as the main spread of disease in the groves studied.

A Statistical Evaluation of Methods of In-Vitro Growth Assessment for Phyllosticta citricarpa: Average Colony Diameter vs. Area

Fungal growth inhibition on solid media has been historically measured and calculated based on the average of perpendicular diameter measurements of growth on fungicide amended media. We investigated the sensitivity of the calculated area (DA) and the measured area (MA) for assessing fungicide growth inhibition of the ascomycete, Phyllosticta citricarpa on solid media. Both the calculated, DA and the actual measured area, MA were adequate for distinguishing significant treatment effects of fungicide on fungal growth, however MA was more sensitive at identifying significant differences between the controls and fungicide concentrations below 5 ppm.