Jill A. Jenkins

Nuclear Transfer of Synchronized African Wild Cat Somatic Cells into Enucleated Domestic Cat Oocytes

Abstract The African wild cat is one of the smallest wild cats and its future is threatened by hybridization with domestic cats. Nuclear transfer, a valuable tool for retaining genetic variability, offers the possibility of species continuation rather than extinction. The aim of this study was to investigate the ability of somatic cell nuclei of the African wild cat (AWC) to dedifferentiate within domestic cat (DSH) cytoplasts and to support early development after nuclear transplantation. In experiment 1, distributions of AWC and DSH fibroblasts in each cell-cycle phase were assessed by flow cytometry using cells cultured to confluency and disaggregated with pronase, trypsin, or mechanical separation. Trypsin (89.0%) and pronase (93.0%) yielded higher proportions of AWC nuclei in the G0/G1 phase than mechanical separation (82.0%). In contrast, mechanical separation yielded higher percentages of DSH nuclei in the G0/G1 phase (86.6%) than pronase (79.7%) or trypsin (74.2%) treatments. In both species, pronase induced less DNA damage than trypsin. In experiment 2, the effects of serum starvation, culture to confluency, and exposure to roscovitine on the distribution of AWC and DSH fibroblasts in various phases of the cell cycle were determined. Flow cytometry analyses revealed that the dynamics of the cell cycle varied as culture conditions were modified. Specifically, a higher percentage of AWC and DSH nuclei were in the G0/G1 phase after cells were serum starved (83% vs. 96%) than were present in cycling cells (50% vs. 64%), after contact inhibition (61% vs. 88%), or after roscovitine (56% vs. 84%) treatment, respectively. In experiment 3, we evaluated the effects of cell synchronization and oocyte maturation (in vivo vs. in vitro) on the reconstruction and development of AWC-DSH- and DSH-DSH-cloned embryos. The method of cell synchronization did not affect the fusion and cleavage rate because only a slightly higher percentage of fused couplets cleaved when donor nuclei were synchronized by serum starvation (83.0%) than after roscovitine (80.0%) or contact-inhibition (80.0%). The fusion efficiency of in vivo and in vitro matured oocytes used as recipient cytoplasts of AWC donor nuclei (86.6% vs. 85.2%) was similar to the rates obtained with DSH donor nuclei, 83.7% vs. 73.0%, respectively. The only significant effect of source of donor nucleus (AWC vs. DSH) was on the rate of blastocyst formation in vitro. A higher percentage of the embryos derived from AWC nuclei developed to the blastocyst stage than did embryos produced from DSH nuclei, 24.2% vs. 3.3%, respectively (P < 0.05). In experiment 4, the effect of calcium in the fusion medium on induction of oocyte activation and development of AWC-DSH-cloned embryos was determined. The presence of calcium in the fusion medium induced a high incidence of cleavage of DSH oocytes (54.3%), while oocyte cleavage frequency was much lower in the absence of calcium (16.6%). The presence or absence of calcium in the fusion medium did not affect the fusion, cleavage, and blastocyst development of AWC-DSH-cloned embryos. In experiment 5, AWC-DSH-cloned embryos were transferred to the uteri of 11 synchronized domestic cat recipients on Day 6 or 7 after oocyte aspiration. Recipients were assessed by ultrasonography on Day 21 postovulation, but no pregnancies were observed. In the present study, after NT, AWC donor nuclei were able to dedifferentiate in DSH cytoplasts and support high rates of blastocyst development in vitro. Incomplete reprogramming of the differentiated nucleus may be a major constraint to the in vivo developmental potential of the embryos.

Reproductive endocrine disruption in smallmouth bass (Micropterus dolomieu) in the Potomac River basin: spatial and temporal comparisons of biological effects

A high prevalence of intersex or testicular oocytes (TO) in male smallmouth bass within the Potomac River drainage has raised concerns as to the health of the river. Studies were conducted to document biomarker responses both temporally and spatially to better understand the influence of normal physiological cycles, as well as water quality and land-use influences. Smallmouth bass were collected over a 2-year period from three tributaries of the Potomac River: the Shenandoah River, the South Branch Potomac and Conococheague Creek, and an out-of-basin reference site on the Gauley River. The prevalence of TO varied seasonally with the lowest prevalence observed in July, post-spawn. Reproductive maturity and/or lack of spawning the previous spring, as well as land-use practices such as application of manure and pesticides, may influence the seasonal observations. Annual, seasonal, and site differences were also observed in the percentage of males with measurable concentrations of plasma vitellogenin, mean concentration of plasma vitellogenin in females, and plasma concentrations of 17β-estradiol and testosterone in both sexes. Bass collected in the South Branch Potomac (moderate to high prevalence of TO) had less sperm per testes mass with a lower percentage of those sperm being motile when compared to those from the Gauley River (low prevalence of TO). An inverse relationship was noted between TO severity and sperm motility. An association between TO severity and wastewater treatment plant flow, percent of agriculture, total number of animal feeding operations, the number of poultry houses, and animal density within the catchment was observed.

Flow cytometric evaluation of antibiotic effects on viability and mitochondrial function of refrigerated spermatozoa of Nile tilapia.

Improved techniques for storage and evaluation of fish sperm would enhance breeding programs around the world. The goal of this study was to test the effect of antibiotics on refrigerated sperm from Nile tilapia (Oreochromis niloticus) by use of flow cytometry with 2 dual-staining protocols for objective assessment of sperm quality. Concentrations of 1 x 10(9). sperm/mL were suspended in Ringers buffer at 318 mOsmol/kg (pH 8.0). The fluorescent stains Sybr 14 (10 microM), propidium iodide (2.4 mM), and rhodamine 123 (0.13 microM) were used to assess cell viability and mitochondrial function. Three concentrations of ampicillin, gentamicin, and an antibiotic/antimycotic solution were added to fresh spermatozoa. Motility estimates and flow cytometry measurements were made daily during 7 d of refrigerated storage (4 degrees C). The highest concentrations of gentamicin and antibiotic/antimycotic and all 3 concentrations of ampicillin significantly reduced sperm viability. The highest of each of the 3 antibiotic concentrations significantly reduced mitochondrial function. This study demonstrates that objective sperm quality assessments can be made using flow cytometry and that addition of antibiotics at appropriate concentrations can lengthen refrigerated storage time for tilapia spermatozoa. With minor modifications, these protocols can be adapted for use with sperm from other species and with other tissue types.

Derivation of cat embryonic stem-like cells from in vitro-produced blastocysts on homologous and heterologous feeder cells

The domestic cat is a focal mammalian species that is used as a model for developing assisted reproductive technologies for preserving endangered cats and for studying human diseases. The generation of stable characterized cat embryonic stem cells (ESC) lines to use as donor nuclei may help to improve the efficiency of interspecies somatic cell nuclear transfer for preserving endangered cats and allow the creation of knockout cell lines to generate knockout cats for studying function of specific genes related to human diseases. It will also enable the possibility of producing gametes in vitro from ESC of endangered cats. In the present study, we report the generation of cat embryonic stem-like (cESL) cells from blastocysts derived entirely in vitro. We generated 32 cESL cell lines from 331 in vitro derived blastocysts from which inner cell masses were isolated by immunosurgery or by a mechanical method. Inhibition of cat dermal fibroblast (CDF) proliferation after exposure to mitomycin-C was both dose and time dependent, where doses of 30 to 40 microg/mL for 5 h were most efficient. These dosages were higher than that required to inhibit cell proliferation of mouse fetal fibroblasts (MFF; 10 microg/mL for 2.5 h). Mitomycin-C did not significantly increase necrosis of cells from either species, and had an anti-proliferative effect at concentrations below cytotoxicity. A clear species-specific relationship between feeder layers and derivation of cESL cell lines was observed, where higher numbers of cESL cell lines were generated on homologous cat feeder layers (n = 26) than from those derived on heterologous mouse feeder layers (n = 6). Three cESL cell lines generated from immunosurgery and cultured on CDF maintained self-renewal and were morphologically undifferentiated for nine and twelve passages (69-102 days). These lines showed a tightly packed dome shaped morphology, exhibited alkaline phosphatase activity and immuno-expression of the pluripotent marker OCT-4 and surface marker SSEA-1. Primary colonies at P0 to P3 and cat blastocysts expressed transcription factors OCT-4, NANOG and SOX-2 and the proto-oncogene C-MYC. However, expression was at levels significantly lower than in vitro produced blastocysts. During culture, cESL colonies spontaneously differentiated into fibroblasts, cardiomyocytes, and embryoid bodies. Development of techniques to prevent differentiation of cESL cells will be essential for maintaining defined cell lines.

CRYOPRESERVATION OF SPERM OF RED ABALONE (HALIOTIS RUFESCENS)

Abstract Abalone culture, a developing industry in Baja California, Mexico, would benefit from genetic improvement and controlled breeding. The use of cryopreserved sperm would allow germplasm availability, and this study was designed to develop sperm cryopreservation protocols for red abalone Haliotis rufescens. The acute toxic effects of the cryoprotectants dimethyl sulfoxide (DMSO), propylene glycol (PG), and glycerol (GLY) were assessed after suspending sperm in different concentrations, whereby cryoprotectant treatments of 10% DMSO and 10% GLY equilibrated for 10 min yielded the highest range of motile sperm in preliminary freezing trials and were used for cryopreservation studies. To determine effective cooling rates, three freezing chambers were tested. Replicate samples of sperm from 4 males were placed in 0.5-mL French straws and frozen using a commercial freezing chamber (CFC) used for bull sperm, a programmable rate chamber (PRC), and a manually controlled styrofoam chamber (MCC). For the CFC, the cooling rate was 16°C/min, from 4°C to −140°C. For the PRC and MCC, it was 1°C/min, from −20°C to −30°C. The samples were held at −30°C for 5 min before being plunged into liquid nitrogen (−196°C) for storage, and each sample was thawed in a water bath at 45°C for 8 s. The quality of thawed sperm was determined by estimating percent motility, evaluating membrane integrity using a dual-staining technique and flow cytometry, and estimating fertilization rate. Statistical analyses were performed using 2-way ANOVA where chamber and treatment were the independent variables. Sperm quality parameters were independent. For motilities, a significant interaction was noted between the cryoprotective treatment and the chamber type, whereby motilities for DMSO and GLY were higher (P = 0.0055) using MCC. Membrane integrities were significantly lower after using the PRC than the CFC or the MCC (P = 0.0167). The highest post-thaw motility (48 ± 7%) was found using sperm suspended in 10% glycerol and frozen in the MCC. The highest percent of intact membranes (56 ± 11%) was for sperm suspended in 10% glycerol and frozen in the CFC. The highest fertilization rate (29 ± 10%) was with samples frozen with 10% glycerol in the CFC. The use of cryopreserved sperm from red abalone provides an alternative breeding option for culture and the protocols delineated are the first developed for this species.

Reproductive health of yellow perch Perca flavescens in selected tributaries of the Chesapeake Bay

Reduced recruitment of yellow perch has been noted for a number of years in certain urbanized watersheds (South and Severn Rivers) of the Chesapeake Bay. Other rapidly developing watersheds such as Mattawoman Creek are more recently showing evidence of reduced recruitment of anadromous fishes. In this study, we used a battery of biomarkers to better document the reproductive health of adult yellow perch collected during spring spawning in 2007-2009. Perch were collected in the South and Severn Rivers, Mattawoman Creek and the less developed Choptank and Allens Fresh watersheds for comparison. Gonadosomatic indices, plasma reproductive hormone concentrations, plasma vitellogenin concentrations and gonad histology were evaluated in mature perch of both sexes. In addition, sperm quantity (cell counts) and quality (total and progressive motility, spermatogenic stage and DNA integrity), were measured in male perch. Many of these biomarkers varied annually and spatially, with some interesting statistical results and trends. Male perch from the Choptank and Allens Fresh had generally higher sperm counts. In 2008 counts were significantly lower in the perch from the Severn when compared to other sites. The major microscopic gonadal abnormality in males was the proliferation of putative Leydig cells, observed in testes from Severn and less commonly, Mattawoman Creek perch. Observations that could significantly impact egg viability were an apparent lack of final maturation, abnormal yolk and thin, irregular zona pellucida. These were observed primarily in ovaries from Severn, South and less commonly Mattawoman Creek perch. The potential association of these observations with urbanization, impervious surface and chemical contaminants is discussed.

Use of Eyeballs for Establishing Ploidy of Asian Carp

Abstract Grass carp Ctenopharyngodon idella, silver carp Hypophthalmichthys molitrix, and bighead carp H. nobilis are now established and relatively common in the Mississippi and Atchafalaya rivers. Commercial fishers of Louisianas large rivers report recurrent catches of grass carp, and the frequency of bighead carp and silver carp catch is increasing. Twelve black carp Mylopharyngodon piceus were recently captured from the Mississippi and Atchafalaya River system, and 10 were analyzed for ploidy. By using the methods described herein, all 10 fish were determined to be diploid. Such correct identifications of ploidy of feral Asian carp species, as well as other species, would provide science-based information constructive for meeting reporting requirements, tracking fish movements, and forecasting expansion of species distribution. To investigate the postmortem period for sample collection and to lessen demands on field operations for obtaining samples, a laboratory study was performed to determine the ...

Novel associations between contaminant body burdens and biomarkers of reproductive condition in male Common Carp along multiple gradients of contaminant exposure in Lake Mead National Recreation Area, USA

Adult male Common Carp were sampled in 2007/08 over a full reproductive cycle at Lake Mead National Recreation Area. Sites sampled included a stream dominated by treated wastewater effluent, a lake basin receiving the streamflow, an upstream lake basin (reference), and a site below Hoover Dam. Individual body burdens for 252 contaminants were measured, and biological variables assessed included physiological [plasma vitellogenin (VTG), estradiol-17β (E2), 11-ketotestosterone (11KT)] and organ [gonadosomatic index (GSI)] endpoints. Patterns in contaminant composition and biological condition were determined by Principal Component Analysis, and their associations modeled by Principal Component Regression. Three spatially distinct but temporally stable gradients of contaminant distribution were recognized: a contaminant mixture typical of wastewaters (PBDEs, methyl triclosan, galaxolide), PCBs, and DDTs. Two spatiotemporally variable patterns of biological condition were recognized: a primary pattern consisting of reproductive condition variables (11KT, E2, GSI), and a secondary pattern including general condition traits (condition factor, hematocrit, fork length). VTG was low in all fish, indicating low estrogenic activity of water at all sites. Wastewater contaminants associated negatively with GSI, 11KT and E2; PCBs associated negatively with GSI and 11KT; and DDTs associated positively with GSI and 11KT. Regression of GSI on sex steroids revealed a novel, nonlinear association between these variables. Inclusion of sex steroids in the GSI regression on contaminants rendered wastewater contaminants nonsignificant in the model and reduced the influence of PCBs and DDTs. Thus, the influence of contaminants on GSI may have been partially driven by organismal modes-of-action that include changes in sex steroid production. The positive association of DDTs with 11KT and GSI suggests that lifetime, sub-lethal exposures to DDTs have effects on male carp opposite of those reported by studies where exposure concentrations were relatively high. Lastly, this study highlighted advantages of multivariate/multiple regression approaches for exploring associations between complex contaminant mixtures and gradients and reproductive condition in wild fishes.

Cloned embryos from semen. Part 2: Intergeneric nuclear transfer of semen-derived eland (Taurotragus oryx) epithelial cells into bovine oocytes

The production of cloned offspring by nuclear transfer (NT) of semen-derived somatic cells holds considerable potential for the incorporation of novel genes into endangered species populations. Because oocytes from endangered species are scarce, domestic species oocytes are often used as cytoplasts for interspecies NT. In the present study, epithelial cells isolated from eland semen were used for intergeneric transfer (IgNT) into enucleated bovine oocytes and compared with bovine NT embryos. Cleavage rates of bovine NT and eland IgNT embryos were similar (80 vs. 83%, respectively; p > 0.05); however, development to the morula and blastocyst stage was higher for bovine NT embryos (38 and 21%, respectively; p < 0.0001), than for eland IgNT embryos (0.5 and 0%, respectively). DNA synthesis was not observed in either bovine NT or eland IgNT cybrids before activation, but in 75 and 70% of bovine NT and eland igNT embryos, respectively, cell-cycle resumption was observed at 16 h postactivation (hpa). For eland IgNT embryos, 13% had > or = 8 cells at 84 hpa, while 32% of the bovine NT embryos had > or = 8 cells at the same interval. However, 100 and 66% of bovine NT and eland IgNT embryos, respectively, that had > or = 8 cells synthesized DNA. From these results we concluded that (1) semen-derived epithelial cell nuclei can interact and be transcriptionally controlled by bovine cytoplast, (2) the first cell-cycle occurred in IgNT embryos, (3) a high frequency of developmental arrest occurs before the eight-cell stage in IgNT embryos, and (4) IgNT embryos that progress through the early cleavage stage arrest can (a) synthesize DNA, (b) progress through subsequent cell cycles, and (c) may have the potential to develop further.

Immediate and delayed (after cooling) effects of centrifugation on equine sperm

The objectives of this study were to determine the effects of centrifugation on equine sperm total and progressive motility, viability, and acrosomal integrity. We hypothesized that although high centrifugation forces would be detrimental to equine Equus caballus sperm, recovery rates would increase. Ejaculates from six stallions were collected, extended to a concentration of 25x10(6) cells/mL, and subjected for 10min to (1) no centrifugation (NC) or (2) centrifugation at 400xg, (3) 900xg, or (4) 4500xg. Before and after centrifugation (Day 0), and after 24h of cooling (Day 1), sperm motility was assessed by computer-assisted semen analysis, and samples were stained with SYBR-14/propidium iodide (PI) for viability and with PI/fluorescein isothiocyanate (FITC)-Peanut aglutinin (PNA) (Arachis hypogaea) for acrosomal integrity. The effect of treatment and day on motility, viability, and acrosomal integrity was determined using a mixed linear model. Compared with the other treatments, centrifugation at 4500xg reduced all end points measured (P<0.05). Both 400xg and 900xg yielded lower recovery rates than that of 4500xg (NC=100.0+/-0.0%; 400xg=54.4+/-8.6%; 900xg=75.0+/-7.1%; 4500xg=97.9+/-2.8%; P<0.05). Centrifugation at 400xg or 900xg did not damage equine sperm. Based on these findings, further studies of centrifugal forces between 900xg and 4500xg are warranted to determine the optimal force that maximizes recovery rate, minimizes sperm damage, and does not affect fertility.