The conservation performance of protected areas (PAs) is typically measured according to whether species are likely to be present within PAs. However, to attain the goal of long-term conservation it is important to consider the performance of PAs in terms of their ability to support the persistence of the species they contain. We used the concept of minimum viable population (MVP) size to examine the potential for PAs within a large national PA network to support mammal species over the long term. By developing habitat models for each species and estimating the area required to support the MVP size for each species, we identified whether each PA had sufficient habitat to meet the species’ requirements. We found that as a whole the PA network is able to support at least one viable population for all species studied. However, the extent of protection offered to species by the PA network varies considerably, with many PAs not able to support viable populations of individual species on their own. By understanding the capacity of PAs to provide long-term protection to species, our findings can guide strategies to increase the resilience of PA networks as a whole, including by improving habitat quality within and connectivity between PAs.

Given the importance of angiostrongyliasis as an emerging infectious disease of humans, companion animals, and wildlife, the current study focused on the transmission dynamics of first- and third-stage larvae of the parasitic nematode, Angiostrongylus cantonensis. The migration of infective larvae and their subsequent distribution within the Lymnaeidae snail, Bullastra lessoni, were investigated over time using microscopic examination of histological sections and fresh tissue. Snails were divided into four anatomical regions: (i) anterior and (ii) posterior cephalopedal masses, (iii) mantle skirt and (iv) visceral mass. The viability of free-swimming third-stage larvae, after their release from snail tissues, was evaluated in vitro by propidium iodide staining and infectivity by in vivo infection of Wistar rats. Snails were sequentially dissected over time to assess the number and anatomical distribution of larvae within each snail and hence infer their migration pathway. Herein, ongoing larval migratory activity was detected over 28 days post-infection. A comparison of infection rates and the larval distribution within the four designated snail regions demonstrated a significant relationship between anatomical region and density of infective larvae, with larvae mostly distributed in the anterior cephalopedal mass (43.6 ± 10.8%) and the mantle skirt (33.0 ± 8.8%). Propidium iodide staining showed that free-swimming third-stage larvae retained viability for between 4 and 8 weeks when stored under laboratory conditions. In contrast to viability, larval infectivity in rats remained for up to 2 weeks only. Knowledge gained from the current work could provide information on the development of new approaches to controlling the transmission of this parasite.

This study evaluated the effect of fibroblast growth factor-2 (FGF-2) on the morphology, primordial follicle activation and growth after in vitro culture of domestic cat ovarian tissue. Ovaries (n = 12) from prepubertal domestic cats were collected and fragmented. One fragment was fixed for histological analysis (fresh control). The remaining fragments were incubated in control medium alone or with 10, 50 or 100 ng/ml FGF-2 for 7 days. After in vitro culture, the following endpoints were analyzed: morphology, activation by counting primordial and developing follicles, and growth (follicle and oocyte diameters). Treatment with 100 ng/ml FGF-2 maintained (P > 0.05) the percentage of normal follicles similar to fresh control. Follicle survival was greater (P < 0.05) after culture in 100 ng/ml FGF-2 than in 50 ng/ml FGF-2. The percentage of primordial follicles decreased (P < 0.05) and the percentage of developing follicles increased (P < 0.05) in all treatments compared with fresh tissue. The proportion of developing follicles increased (P < 0.05) in tissues incubated with 100 ng/ml FGF-2 compared with control medium and other FGF-2 concentrations. Furthermore, culture in 10 or 100 ng/ml FGF-2 resulted in increased (P < 0.05) follicle and oocyte diameters compared with fresh tissues and MEM+. In conclusion, FGF-2 at 100 ng/ml maintains follicle survival and promotes the in vitro activation and growth of cat primordial follicles.

Nosema apis and N. ceranae are the two causative agents of Nosema disease in adult honey bees (Apis mellifera L.). Nosema apis has been a recognized parasite for over a century and its epizootiology is well known. In contrast, N. ceranae is an emerging parasite of honey bees, which is now globally prevalent and the dominant Nosema spp. in many parts of the world. Despite this, many gaps in our knowledge exist regarding this species. For example, we do not fully understand all of the routes of transmission of N. ceranae among bees, or how long this parasite is capable of surviving in honey bee colonies. Here we investigated the viability and infectivity of N. ceranae spores in water and 2 M sucrose over time after storage at 33, 20, −12 and −20°C. Spores in both 2 M sucrose and water maintained high viability, except in water at −20°C over the course of the 6-week experiment. Infectivity was variable for spores after storage at all four temperatures, but all were infective at the last time point. The results provide evidence for cold tolerance and suggest that both water and 2 M sucrose (fall bee feed) could act as routes of transmission for N. ceranae. This work also contains information that may help influence management recommendations for the parasite.

This study aimed to evaluate the effects of dexamethasone on development, viability, antrum formation and ultrastructural integrity of bovine secondary follicles cultured in vitro for 18 days. Bovine ovaries were obtained from slaughterhouses and secondary follicles of ~150–200 µm diameter were isolated and cultured in the laboratory in TCM-199+ alone or supplemented with different concentrations of dexamethasone (1, 10, 100 and 1000 ng/ml). Follicle viability was evaluated after the culture period, using calcein-AM (viable) and ethidium homodimer (nonviable). Follicle diameters and antrum formation were evaluated at days 0, 6, 12 and 18. Before or after in vitro culture, follicles were fixed for histological and ultrastructural analysis. Follicle diameters were evaluated using analysis of variance and Kruskal–Wallis test, while chi-squared test was used to evaluate the percentage of viable follicles and antrum formation (P < 0.05). Follicles cultured for 6 days with all treatments increased their diameters significantly, but there was no significant difference between treatments at the end of the culture period. In vitro cultured follicles showed antral cavity formation at the end of the culture period, but no influence of dexamethasone was seen. Ultrastructural analysis showed that follicles cultured with dexamethasone (1, 10, 100 and 1000 ng/ml) had well preserved granulosa cells. However, oocytes from follicles cultured with 10, 100 or 1000 ng/ml dexamethasone showed signs of degeneration. It can be concluded that follicles cultured in vitro in the presence of dexamethasone demonstrated continuous in vitro growth, but oocytes from follicles cultured with 10, 100 or 1000 ng/ml dexamethasone had poor ultrastructure.

Metacercariae of various species within the genus Holostephanus Szidat, 1936 (Trematoda: Digenea: Cyathocotylidae) occur in muscles of both farmed and wild fish, including common carp (Cyprinus carpio Linnaeus, 1758). The life cycle includes a snail as first intermediate host, fish as second intermediate host and birds or mammals as final hosts. We studied the zoonotic potential and the viability of Holostephanus metacercariae from common carp following exposure to various physical and chemical treatments. Muscle tissue samples of common carp specimens from a fish farm in the north-eastern part of Hungary were examined and metacercariae recovered. The zoonotic potential was evaluated experimentally by using small mammals as models (albino mice, n = 2; and Syrian hamsters, n = 4) infected per os with Holostephanus cysts. Parallelly, Metagonimus metacercariae were used as positive controls. We could not confirm the zoonotic potential of Holostephanus metacercariae as they did not survive in the mammalian intestine whereas Metagonimus metacercariae developed to the adult stage. We assessed the viability of metacercariae isolated from common carp specimens during exposure to different physical treatments (temperatures of −18°C, +20°C, +40°C and +60°C) and chemical agents (5% and 10% acetic acid and 10% sodium chloride (NaCl)). Metacercariae lost viability by freezing at −18°C (2 h), heating at 60°C (20 min), incubation in 5% and 10% acetic acid (5 min) and 10% NaCl (2 h). These methods served as models to investigate the effectiveness of food preparation techniques (such as cold and hot smoking, freezing, salting and pickling) on the survival of metacercariae.

This study aimed to compare three methods of cell death assessment [trypan blue exclusion (TBE), propidium iodide viability assay (PIVA), and transmission electron microscopy] to evaluate fresh and frozen–thawed chicken primordial germ cells (PGCs). For this study, chicken PGCs were collected from ROSS 908 and Oravka breed hens, cryopreserved-thawed according to the protocol, and submitted for different cell death assessments. We observed significant differences between TBE and PIVA techniques in the detectable proportion of dead cells in fresh (14.14 ± 1.27 versus 7.16 ± 1.02%, respectively) and frozen–thawed (44.00 ± 2.11 versus 33.33 ± 1.67%, respectively) samples of the Oravka breed. Moreover, significant differences (p < 0.05) between TBE and PIVA techniques in the detectable proportion of dead cells in fresh (9.20 ± 0.60 versus 5.37 ± 0.51%) samples of ROSS 908 breed were recorded. Differences may be due to methodological, sensitivity, and toxicity features of each technique tested, where TB stains cell cytoplasm of dead cells and PI penetrates and intercalates into DNA of dead cells. Therefore, we suggest using a more precise and sensitive PIVA for viability evaluation of PGCs. Further research is needed to apply various fluorochromes for more detailed cell viability evaluation.

The present study determined the prevalence of hydatid cysts in different organs of slaughtered hilly ‘Gaddi’ breed small ruminants—sheep (n = 230) and goats (n = 197)—in Kangra Valley of the north-western Himalayas, India. Hydatid cysts were found in 12.2% (n = 28) of sheep and 10.7% (n = 21) of goats. Pulmonary echinococcosis was more prevalent in slaughtered sheep and goats (sheep 56.36%; goats 62.90%) than hepatic echinococcosis (sheep 43.64%; goats 37.10%). Fertility rates were higher in hepatic (81.25%) and pulmonary cysts of sheep (83.87%) compared to goats. Molecular identification and genotypic characterization of Echinococcus granulosus isolates were based on mitochondrial cytochrome oxidase 1 gene (mtCO1). The genotypic characterization identified the isolated strain to be closely related to the G7 genotype. Histopathological examination revealed a thick coat of granulation tissue, causing fibrosis and inflammatory reaction composed of fibroblasts and mononuclear cells around the cysts. In the liver, hepato-cellular degeneration was prominent at the periphery of the cysts. The present study highlights the molecular confirmation and phylogenetic analysis of E. granulosus isolates with the prevalence of hydatidosis in a naïve host species and in an unexplored region. The findings are of significant medical and veterinary importance regarding development of control measures to check dissemination of hydatidosis.

In vitro maintenance of helminth parasites enables a variety of molecular, pharmaceutical and immunological analyses. Currently, the nutritional and environmental in vitro requirements of the equine ascarid parasite, Parascaris spp., have not been determined. Additionally, an objective method for assessing viability of Parascaris spp. intestinal stages does not exist. The purpose of this study was to ascertain the in vitro requirements of intestinal stages of Parascaris spp., and to develop a viability assessment method. A total of 1045 worms were maintained in a total of 212 cultures. Worms obtained from naturally infected foals at necropsy were immediately placed in culture flasks containing 200 mL of culture media. A variety of media types, nutrient supplementation and environmental conditions were examined. A motility-based scoring system was used to assess worm viability. Worms maintained in Roswell Park Memorial Institute-1640 had significantly better viability than any other media (P < 0.0001) and all media types supplemented with any of the nutrients examined (P < 0.0001). The use of a platform rocker also significantly improved viability (P = 0.0305). This is the first study to examine the requirements for maintaining Parascaris spp. intestinal stages in vitro and to evaluate their viability based on movement using an objective scoring system.

Halloysite clay nanotubes are safe and biocompatible nanomaterials and their application in biomaterials is very promising. The microencapsulation of yeast cells in the shell of clay nanotubes modifying their properties was demonstrated here. Each cell was coated with a 200–300 nm-thick tube shell and this coating was not harmful for these cells’ reproduction. Synthesis of magnetic nanoparticles on the surfaces of the nanotubes allowed for magnetic-field manipulation of the coated cells, including their separation. Providing nano-designed shells for biological cells is a step forward in development of ‘cyborg’ microorganisms combining their intrinsic properties with functions added through nano-engineering.

The purpose of this study was to evaluate the effects of FSH and PI3K on the nuclear maturation, viability, steroidogenesis and embryo development of bovine cumulus–oocyte complexes (COCs). Oocyte maturation was achieved with MIV B, MIV B+100 µM LY294002, MIV B+10 ng/mL follicle stimulating hormone (FSH), or MIV B+10 ng/mL FSH+100 µM LY294002 treatments for 22–24 h. After the cultured COCs were denuded, oocytes were separated into those that extruded polar bodies (mature) and those that did not, and real-time polymerase chain reaction (PCR) for BAX, BCL2, LHR, FSHR, CYP11A1, CYP19A1 and HSD17B1 genes was performed. The culture medium was collected to determine the levels of 17β-estradiol (E2) and progesterone (P4). The trypan blue test was used to study COC viability, and embryo development was evaluated. FSH increased nuclear maturation and PI3K blocked the maturation but did not influence oocyte viability. BAX and BCL2 expression levels in the cumulus cells were only affected by FSH, and the BAX levels decreased after treatment with LY294002. FSH increased the levels of E2 and P4, however inhibition of PI3K decreased E2 levels. MIV B enhanced levels of LHR, FSHR, CYP11A1, CYP19A1 and HSD17B1, whereas LY294002 inhibited the expression levels of all genes. MIV B+FSH decreased the expression levels of all genes except CYP11A1. LY294002 did not demonstrate any effects in the presence of FSH. Embryo development was significantly decreased when the MIV B+FSH medium was used. In conclusion, FSH controls the steroidogenesis, viability and gene expression in COCs. PI3K plays essential roles in nuclear maturation, steroidogenesis and embryo development.

We aimed to compare the effect of three different permeating cryoprotectants on the post-thaw spermatozoa quality. Pooled semen from Oravka cock line (n = 6) was diluted in Kobidil+ extender and frozen in cryoprotectant solutions containing 8% dimethylsulfoxide (DMSO), 8% ethylene glycol (EG) or 8% glycerol (GL) in liquid nitrogen vapours before being plunged into the liquid nitrogen. Spermatozoa motility parameters were assessed in vitro after freezing–thawing by a computer-assisted semen analysis (CASA) system and viability status was examined using fluorescent probes. The lower percentage (P < 0.05) of motile and progressively moving spermatozoa immediately after thawing were obtained in all experimental groups (DMSO, EG, GL) compared with the control. Significant (P < 0.05) differences in total motility and progressive movement between GL and DMSO, EG groups were observed. However, the higher number (P < 0.05) of acrosome damaged spermatozoa was found in the DMSO and EG groups and no significant differences were observed in the GL group compared with the control. Differences (P < 0.05) between experimental groups and the control in the results of spermatozoa necrosis were observed. No significant differences in the percentage of apoptotic spermatozoa were found between control and experimental groups. However, significant differences (P < 0.05) in number of live and necrotic spermatozoa between GL and DMSO, EG groups were examined. The findings of the present study indicate that glycerol seems to be suitable for semen cryopreservation in the gene banks. In addition, fertility evaluation in vivo is needed in order to evaluate the possible contribution for the bank of animal genetic resources.

The present study investigated whether dietary turmeric (Curcuma longa L.) can improve rabbit reproduction, ovarian function, growth, or viability. Female New Zealand White rabbits were either fed a standard diet (n=15) or a diet enriched with 5 g (group E1) or 20 g (group E2) turmeric powder per 100 kg feed mixture (n=16 or 15, respectively). After 295 days, weight gain, conception and kindling rates, pup and mother viability, ovarian macro- and micro-morphometric indices, release of leptin in response to the addition LH, and the release of progesterone, testosterone and leptin by isolated ovarian fragments were analyzed. Dietary turmeric failed to affect ovarian length and weight but did increase the number of primary follicles (E2: 32.5% greater than control group), as well as the diameter of primary (E1: +19.4%, E2: +21.1%), secondary (E2: +41.4%), and tertiary (E1: +97.1%, E2: +205.1%) follicles. Turmeric also increased the number of liveborn (E1: +21.0%) and weaned (E1: +25.0%) pups and decreased the number of stillborn pups (E2: −87.5%) but did not affect weight gain, conception, or kindling rate. Furthermore, dietary turmeric decreased doe mortality during the first reproductive cycle (13.3% in control; 0% in E1; and 6.7% in E2) but not during the second cycle. In vitro, the ovaries of the turmeric-treated rabbits released more progesterone (E1: +85.7%, E2: +90.0%) and less testosterone (E2: −87.0%) and leptin (E2: −29.0%) than the ovaries of control rabbits. Moreover, LH decreased the leptin output of control rabbits but increased that of experimental rabbits. Therefore, it is likely that dietary turmeric improves pup viability and that it could promote rabbit fecundity by either (1) promoting the production of primary ovarian follicles or (2) stimulating the growth of follicles at all stages of folliculogenesis.

Weed seed viability is an important parameter to assess the efficacy of soil disinfestation methods like fumigation and steam. In field experiments, seed samples are commonly placed in permeable bags and buried at several depths in soil before the application of soil disinfestation treatments. The seed samples are recovered several days to weeks after treatment and then seed viability is determined in the laboratory. The process of sample installation and recovery is time consuming and may expose personnel to hazardous conditions such as heat or fumigants. Described is a custom soil probe system, developed to simplify installation and recovery of weed seeds from soil. Each soil probe is capable of holding weed seed samples at three different depths up to 30 cm. The following hypothesis was tested: viability of weed seeds is similarly affected by soil disinfestation treatments whether the seeds were contained in the soil probe system or seed bag assays. Two different soil disinfestation trials were conducted: (1) a repeated micro-plot study (USDA Salinas, 1 m-2), using steam as a soil disinfestation treatment and (2) a field study in a commercial strawberry field with 1,3-dicloropropene plus chloropicrin (Pic-Clor 60) as soil disinfestation method. In both studies, seed viability of burning nettle, common knotweed, and common purslane (tetrazolium assay) and germination rates of yellow nutsedge tubers were assessed. Results indicate that the soil probe system can be used as an alternative to the seed bag assay to assess weed control efficacy of described soil disinfestation methods.

Changes in dormancy and viability of freshly harvested seed of yellow nutsedge buried in the field, at depths of 5, 25, and 50 mm, were studied over a 2-yr period. The number of dormant, viable seed decreased most rapidly at 5 mm because more seed lost their dormancy and germinated (up to 30%) than at lower depths. Loss of viability through decay of seed appeared similar at all depths. No seedlings emerged from any depth. In a laboratory experiment, conducted in optimal soil moisture conditions, seedlings emerged from all seed that germinated, demonstrating that adequate soil moisture was critical for seedling establishment. This was substantiated in a second field experiment in which irrigation and mulching greatly increased seedling survival. The number of seedlings surviving in irrigated plots was 0.78% of seed sown and 0.03% in the rain-fed plots. The role of seed in the establishment of yellow nutsedge infestations is probably of little importance in dryland cropping areas despite the longevity and viability of the seed.

Seed dormancy does not play a role in the germination ecology of curly dock (Rumex crispus L. ♯ RUMCR). This study confirms reports that freshly matured seeds are nondormant, and it shows that buried seeds exposed to natural seasonal temperature changes remain nondormant. From October 1981 through June 1983, seeds exhumed at monthly intervals germinated 80 to 100% at all thermo-periods. These results do not support suggestions that seeds of curly dock buried in soil enter dormancy. However, the results do explain why seeds of this species in the Beal and Duvel buried-seed experiments germinated when exhumed at various times during the growing season.

The aim of this work was to examine the influence of cryostorage duration of Pinzgau bull's insemination doses (IDs) on some sperm traits. The IDs were frozen by a slow freezing method and stored in liquid nitrogen for different periods: less than 8 years (group 1), 8–13 years (group 2) and 14–18 years (group 3). Motility (CASA), pathological sperm rate (Giemsa staining), apoptotic (Yo-Pro-1-positive) and necrotic (propidium iodide-positive) cell occurrence and fertilizing ability (penetration/fertilization test) of spermatozoa were evaluated post-thaw. The average post-thaw sperm motility in all examined groups was over 40%. No significant influence of storage length either on the sperm total motility or progressive movement was revealed. In each tested group the average rate of malformed spermatozoa did not exceed 20%. No effect of cryostorage length on the occurrence of apoptotic or necrotic sperm was noted. Similarly, penetrating/fertilizing ability of sperm did not differ among the groups, excepting differences in the rate of pronuclei (PN) formation. In group 1, 72.9% of eggs showed two visible PN following 20 h incubation with sperm, whilst in groups 2 and 3 only 67 and 54.5% of zygotes, respectively, had both PN at this time. These results revealed no influence of storage time on the bull spermatozoa in all parameters excepting the rate of PN formation. As high inter-male variability was observed in the susceptibility of bull sperm to cryostorage, individual differences should be taken into account when semen from individual bulls is to be stored for a long time.

Musk thistle is an invasive weed that is widely distributed throughout much of North America, including grasslands in temperate climates of the midwest USA. A series of laboratory and greenhouse experiments were conducted to determine the effect of various environmental factors on germination of musk thistle seeds. In temperature-fluctuation experiments, seed germination was greater than 65% in both alternating (30/20 C) and constant (20 or 25 C) temperature regimes with an 8-h day but less (33%) in warmer regimes (35/20 C). Germination of musk thistle seeds was 37% in alternating temperature regimes of 30/20 C in total darkness, but less than 67% in pots in the greenhouse. Differences of 10 and 15 C between day and night temperatures resulted in 91 and 75% maximum germination of musk thistle, respectively. Increasingly dryer soils reduced germination of musk thistle seeds from 35% (−0.03 MPa) to 0% (−1.2 MPa), whereas saline soils (> 80 mM) reduced maximum germination to less than 10%. Musk thistle seeds collected from populations in a bare-ground area had 96% germination, which was greater than that of seeds collected from populations growing in a perennial grass pasture (71%). A residence time (i.e., period that seeds remained on the parent plant) of 9 to 12 wk after capitulum maturity resulted in seeds germinating more quickly than those dispersed earlier. Overall, reduced light levels, cool and fluctuating temperatures, and amount of time seeds remained in residence are some of the most important factors that contribute to germination of musk thistle seeds. Information on germination dynamics of musk thistle seeds provides an understanding of the interactions that affect this process and underscores the importance of timely management strategies in temperate grasslands.

Jubatagrass is one of the most invasive nonnative species along sensitive natural coastal sites of California. This study was designed to understand the biology of reproduction and seed longevity under field conditions. Jubatagrass can produce over 100,000 wind-dispersed seeds from a single inflorescence. Seeds are produced apomictically, and germination is directly related to seed size. Of the total seeds produced, only 20 to 30% were of ample size to readily germinate when exposed to light and under a temperature range similar to coastal environments. Seeds not exposed to light also germinated but at about 30% the level of light-exposed seeds. This suggests that exposed disturbed coastal sites with moderate temperatures have high potential for germination and establishment of jubatagrass. The percentages of germinable and viable seeds were not significantly different, indicating that jubatagrass does not have a primary dormancy. This was supported by field experiments demonstrating that seeds do not persist under natural conditions for more than 6 mo. These results indicate that an intensive 1-yr control program targeting established seedlings and mature plants should sufficiently manage existing populations. However, effective long-term management of jubatagrass must focus on anticipating environments susceptible to invasion, reducing new seed recruitment, and preventing subsequent seed germination and seedling establishment.