Showing papers in "Virginia journal of science in 2008"

Dinoflagellate Cysts Within Sediment Collections From the Southern Chesapeake Bay, and Tidal Regions of the James, York, and Rappahannock Rivers, Virginia

Abstract: The upper 2 cm of sediment core samples from 70 stations in the tidal waters of three Virginia rivers and at 23 stations in the lower Chesapeake Bay were sampled for dinoflagellate cysts. The river sediment cysts were dominated by three common bloom producing species (Heterocapsa triquetra, Scrippsiella trochoidea, and Cochlodinium polykrikoides ), whereas these were in low concentration in the Chesapeake Bay sediments which contained mainly dinoflagellate cysts of neritic and oceanic taxa. The mean sediment concentrations from stations in the James, York, and Rappahannock rivers were respectively 1174.8, 536.2, and 323.6 cysts g1. The mean cyst concentration in the Chesapeake Bay sediment was 714. 8 t 1. Cysts of 2 potentially harmful species were recorded from the sediment, with the river sediments identified as seed beds and a source for re-occurring algal blooms in

Low-Density Rodent Communities in Eastern Virginia

Abstract: Two communities of small mammals were live trapped every other week for 15 months in linear oldfield habitat bordered by forested swampland in eastern Virginia. All nine species of the rodent community were present, mostly in low numbers and often intermittently. All species were characterized by high transiency, with a minority of marked animals becoming resident. Despite high trappability, all but two species in these communities had extremely low densities, suggesting that most species could not sustain populations via in situ reproduction. INTRODUCTION Populations of small mammals usually are studied only at locations where favorable habitat makes the production of moderate-to-high densities a probability in most years, yielding many observations for the amount of expended field work and making statistical testing possible. Similarly, communities of small mammals are studied in the primary habitat of that group, such as grassland, forest, or desert. When a linear habitat of one type passes through a matrix of another habitat type, such as when a powerline, with its open habitat beneath, passes through a forest, the possibilities of a broader, more speciose community of small mammals can be realized. We studied two locations in the Dismal Swamp of eastern Virginia, with the goal of conducting a population study of the Dismal Swamp subspecies of Synaptomys cooperi helaletes, the southern bog lemming. Earlier field studies using pitfall traps (Rose 1981) had revealed its presence, after speculation by several investigators (Handley 1979, Meanley 1973, Taylor 1974), that this arvicoline rodent might be extinct because it had not been found since the U. S. Biological Surveys conducted in the Dismal Swamp in the 1890s (Merriam 1896). The objectives of our study were to: 1. conduct a population study of the southern bog lemming, 2. describe the dynamics of small mammal communities in two clearings of the Dismal Swamp, a forested swampland, and 3. evaluate the spatial distributions of small mammals based on the vegetational structure of the habitat. METHODS Our study was conducted in the 40 m wide opening under a 110 kv powerline in the 1 Corresponding author: Robert K. Rose, Department of Biological Science, Old Dominion University, Norfolk, Virginia 23529-0266. 757-343-4409, brose@odu.edu 170 VIRGINIA JOURNAL OF SCIENCE northwest comer of the Great Dismal Swamp National Wildlife Refuge, where the first specimens of Synaptomys cooperi were collected in the 20 Century (Rose 1981 ). The vegetation under the powerline is maintained at an early successional stage by 3-5 y mowings by Dominion Virginia Power. One study grid was placed in an area dominated by American cane (Arundinaria gigantea), the other in an area with a thick herbaceous layer of panic grasses (Panicum spp.) and soft rushes (Juncus spp.). Both grids had numerous scattered seedlings and saplings of red maple (Acer rubrum ), sweet gum (Liquidambar styraciflua) and loblolly pine (Pinus taeda). Our 15 mo of trapping began in December and ended in February of the second year. Grid 1 was 6 X 14 and Grid 2, located 300 m to the east, was 6 X 15. One Fitch live trap (Rose 1973) baited with wildbird seed and sunflower seed was placed at 7 .6 m intervals at the coordinates on the grids. We checked traps for three mornings every two weeks and newly caught small mammals were given uniquely numbered ear tags. Standard information on reproductive condition, body mass, and location was collected for all animals in the manner of Krebs et al. (1969). We released all animals at the point of capture, except for shrews, which usually died in the traps; some rodents ( ca. 5-8) were removed from traps and killed by predators. There was no other trap-induced mortality. Trappability, the proportion of animals captured during a given month known to be alive in that month, was high (>75%), indicating that populations were being adequately censused (Hilborn et al. 1976). Despite high trappability, more than half of tagged rodents were not recaptured, i.e., they were transients. So few were recaptured multiple times that we defined residents as any animal caught in a later trapping period. In the calculation of home range, we used the inclusive boundary strip method of Stickel ( 1954 ), which adds a strip equal to one-half of the inter-trap interval to the smallest area enclosing stations of capture. Thus, the trappable area of Grid 1 was 0.38 ha and that of Grid 2 was 0.40 ha, values that also will be used in the calculation of density. Because small mammals are known to respond to the structural habitat produced by a variety of micro habitat variables (M' Closkey 197 6, Dueser and Shugart 1978), we measured a set of habitat variables on each grid. We sampled the vegetation of both grids at the end of the growing season in September, using a point-intercept method (Muller-Dombois and Ellenberg 1974) with a modified point-frequency frame. Our sampling frame consisted of a 2.1 m long pole attached to a tripod at the center point, with holes placed at 25 cm intervals. We centered the tripod over the trap station and then dropped eight steel rods marked at 10 cm intervals through the holes. We recorded the highest point of contact on each rod for both woody and herbaceous species. We also recorded the number of stem contacts per each 10 cm interval for the rods located 50 cm from the center point. The frame was then rotated 90° and the process repeated. We used this sampling method because the characteristics of each trapping station rather than the generalized structure of the vegetation of the entire grid could be measured. Using these and other measurements (Table 1), we recorded 12 non-redundant habitat variables for each of the 17 4 trapping stations. We selected these variables because they provided vegetative information either known or believed to influence distributions of small mammals (e.g., M'Closkey 1976, Dueser and Shugart 1978). Discriminant function analysis (DFA) of the 12 habitat variables, computed with the discriminant procedures of SPSS (Nie et al. 197 5), was used to describe microhabitat RODENT COMMUNITIES IN VIRGINIA 171 TABLE 1. The 12 habitat variables used for the analysis of habitat structure; the first five were measured or estimated, the remainder determined from vegetation contacts on points of a sampling frame MNEMONIC VARIABLE ASSESSMENT AVVH Average vegetation Estimation of vegetative height in a 1-m radius at the height trap MAXVH Maximum vegetation Estimation of tallest vegetation in a 1-m radius of the height trap HTV Height of tallest Estimation of tallest vegetation in a 2-m radius of the vegetation trap LIT Litter depth Average depth of litter within a 1-m radius of the trap AVF Average distance to Distance from trap to forest edge forest edge STD1 Stem density, first Number of vegetative contacts between O and 20 cm interval from ground surface on the steel rods located 50 cm from the center point of the tripod STD2 Stem density, second Same as STDl, but 21-40 cm from ground surface interval STD3 Stem density, third Same as STD 1, but 41-60 cm from ground surface interval STD4 Stem density, fourth Same as STDl, but 61-80 cm from ground surface interval STD5 Stem density, fifth Same as STD 1, but 81-100 cm from ground surface interval PCW Percent woody % of steel rods of sampling frame with woody vegetation contacts PCH Percent herbaceous Percent herbaceous vegetation vegetation differences in the 10 species of small mammals that were trapped. The mark-recapture data were analyzed using programs developed by Krebs (1999), which determined Minimum Number known to be Alive (MNA), an estimate of population density for each species. Chi-square tests and correlation analysis were used, also with SPSS. RESULTS The small mammal community and population density: With only two southern bog lemmings being tagged on Grid 1 and 11 others on Grid 2 (Fig. 1 ), a population study of Synaptomys cooperi was not possible. Furthermore, lemmings were only present for two months on Grid 1 and for five consecutive months at the end of the study on Grid 2. The highest calculated density on Grid 1 was 5/ha and on Grid 2 ca. 18-20/ha. Relatively little information on

Topographic Factors Affecting the Tree Species Composition of Forests in the Upper Piedmont of Virginia

Abstract: There are many factors that influence forest species composition and many are linked to topographical features. This study, conducted on the Ferrum College campus in the Upper Piedmont Physiographic Province of Virginia revealed three major forest types associated with topographic factors using cluster analysis and detrended correspondence analysis . The first type of forest occurred mostly on northeastern slopes on toe slope topographic positions and was mainly composed of tulip tree (Liriodendron tulipifera) and red maple (Acer rub rum). The second type of forest was found on shoulder and side slope positions and was composed mostly of high densities of sourwood ( Oxydendrum arboreum ), red maple and chestnut oak (Quercus prinus) species. The final forest type was located mostly on ridgetops and shoulder slope positions with a southwestern aspect and was composed mostly of white pine (Pinus strobus), sourwood, chestnut oak and scarlet oak (Quercus coccinea). In general, tree density increased with ascending slope position while DBH decreased. Species richness did not differ significantly by topographic position or aspect. INTRODUCTION There are many variables that influence forest species composition including soil moisture and nutrients, air temperature, light and disturbance regime. These variables are often strongly linked to topographic features such as aspect, slope position, inclination and elevation (Desta et al. 2004). Edaphic and topographic factors exert important influences along the upper Piedmont and Blue Ridge physiographic provinces of Virginia (Stephenson 1982, Harrison et al. 1989, Farrell and Ware 1991, Copenheaver et al. 2006). These forests, however, also have a long and complex disturbance history that has affected forest species composition. The forests in this region of Virginia were once dominated by American chestnut (Castanea dentata) until the invasion of the chestnut blight fungus (Endothia parasitica) in the 1920s (Johnson and Ware 1982). Following this event, highest rankings of density and basal area have been shared by a number of tree species, 1 corresponding author; tfredericksen@ferrum.edu 4 VIRGINIA JOURNAL OF SCIENCE predominately oaks (Quercus) and hickories (Carya) (Johnson and Ware 1982). A wide range of other natural and anthropogenic factors including recent ice storms (Stueve et al. 2007) and gypsy moth defoliation (Whitmire and Tobin 2006) also influence the species composition of Appalachian and Piedmont forests in Virginia. In addition, selective logging, deer browsing and the spread of invasive plant species, particularly ailanthus (Ailanthus altissima), continue to impact the structure and composition of these forests (Carter and Fredericksen 2007). This study characterized the species composition of the forests on the property of Ferrum College located on the Upper Piedmont Physiographic Province close to the Blue Ridge Escarpment in Franklin County, Virginia. Data were collected on topographic position and aspect in order to interpret the species composition in relation to topographic variables. MATERIALS AND METHODS During 2006 and 2007, 19 permanent plots were established in forested areas of the 700-acre Ferrum College campus (N 36.5°, W 80.1°). Plantation forests were not included in this study. Plots were 20 x 20 m in size and were initially established randomly from a topographical map. After selection of the first eight plots, however, an effort was made to select plot locations based on representation of possible aspect and slope position combinations, with each individual slope position and aspect being represented at least twice, except for northwest slope positions which had n = 1. The stands chosen for this study had not been subjected to recent logging; however, all of the stands had most likely been subjected to light selective logging during the 1970s, mostly for oak species and tulip tree (Liriodendron tulipifera ). The elevation of the plots ranged from approximately 300-400 m. Each tree in the plot with diameter at breast height (DBH) of~ 10cm was identified to species, tagged, and evaluated for crown class, crown condition, stem quality and stem condition. Each plot location was marked and recorded with a GPS mapping system, and aspect and slope position in the topography were recorded. Aspect was measured with a compass and slope position was categorically determined as ridge top, slope shoulder, side slope, toe slope, or valley bottom. Average DBH, average basal area, average density and species richness were calculated by site according to aspect and also by slope position. Cluster analysis and detrended correspondence analysis (DCA) were used to determine similarities among plots with respect to species composition. Rare species were down-weighted in the analyses because they can exert an effect on ordinations that is disproportionate to their abundance. All ordination analyses were carried out using PC-ORD (Version 5, MJM Software, G leneden Beach, Oregon). Kruskal-Wallis (K-W) non-parametric tests were conducted to determine if species richness, tree density, or mean tree diameter varied among slope position, aspect, or plot groupings generated by DCA and cluster analysis. Differences were considered statistically significant at p ::_ 0.05. Analyses were carried out using SYST AT 10.2 (SYST AT Software, Inc., San Jose, CA). RESULTS The study plots contained 498 trees and 23 tree species. In these plots, the most abundant species were tulip tree, sourwood (Oxydendrum arboreum), and red maple TOPOGRAPHY AND FOREST COMPOSITION 5 TABLE 1. Number of plots (N), average diameter at breast height (DBH), tree density, species richness, and top three most abundant species by topographic position in forested plots on the property ofFerrum College, Franklin County, VA. Means with the same letter are not significantly different at p :::_ 0.05 . Li Tu = Liriodendron tulipifera, OxAr = Oxydendrum arboreum, AcRu = Acer rubrum, PiSt = Pinus strobus , QuPr = Quercus prinus , QuCo = Quercus coccinea, AiAl = Ailanthus altissima . Topographic N DBH Density Species Most abundant Position (cm) (#/ha) Richness species Valley 2 33 .7 a 238 b 3.0 a LiTu, AcRu, AiAl Toe 4 24 .6 b 600 ab 5.5 a LiTu, AcRu, OxAr Side 5 22 .0 be 725 a 6.4 a LiTu, AcRu, OxAr Shoulder 5 20.0 C 670 ab 5.4 a QuPr, OxAr, PiSt Ridge 3 20.0 C 800 a 5.7 a PiSt, OxAr, QuCo TABLE 2. Number of plots (N), average diameter at breast height (DBH), tree density, species richness, and top three most abundant species by aspect in forested plots on the property of Ferrum College, Franklin County, VA . Means with the same letter are not significantly different at p :::_ 0 .05 . LiTu = Liriodendron tulipifera, OxAr = Oxydendrum arboreum, AcRu = Acer rubrum , PiSt = Pinus strobus , QuPr = Quercus prinus, AiAl = Ailanthus altissima, QuAl = Quercus alba . Aspect N DBH Density Species Most abundant species (cm) (#Iha) Richness NE 7 21.6 a 685 a 6.3 a LiTu, OxAr, AcRu NW 1 22.4 a 650 a 4.0 a OxAr, PiSt, AcRu SE 3 23.4 a 633 a 6.0 a AcRu, LiTu, QuAl SW 3 21.6 a 675 a 5.0 a PiSt, QuPr, OxAr (Acer rub rum). Red maple was found in every plot and sourwood appeared in all but three of the plots. Tulip tree was found in slightly over half of the plots, but it was abundant in the plots in which it was located. White pine (Pinus strobus), and chestnut oak (Quercus prinus) were also common in plots of this study. Some trends were observed with respect to tree density and DBH by the topographic slope classification. In general, tree density increased with ascending topographic position while DBH decreased. Valley plots had a larger mean tree DBH than all other positions (Table 1) and toe slope positions had a significantly higher mean tree DBH than shoulder or ridge top topographic positions. Tree density tended to increase with topographic position (Table 1 ). Species richness did not differ significantly by topographic position (Table 1). No significant differences were observed for aspect in mean tree diameter, species richness, or tree density (Table 2). Cluster analysis (Figure 1 ), revealed three main types of forest tree communities. The attributes of these groups with respect to DBH, tree density, species richness, and 6 VIRGINIA JOURNAL OF SCIENCE p01 p11 pOS p03 pQ6 ---I p04 p07 p09 ~6g----I p12 p18 p1 3 ~1i --I I p19 p15 p16 r-----p02 FIGURE 1. Cluster analysis of the plots within fore sts on the property of Ferrum College , Franklin County, Virginia. The first cluster of plots 1, 11 , 5, 3 and 6 were plots with a modal tendency for toeslope positions and northeastern aspects . Cluster two of plots 4, 7, 8, 9 and 10 represented mostly shoulder and side slope plots. Plots 12-19 made up the third cluster occurring most often on southwestern aspects and ridgetop or shoulder. The main outlier plot identified from the cluster analysis was plot 2, a plot with a very high density (80% ) oftuliptree. TABLE 3. Number of plots (N) , average diameter at breast height (DBH) , tree density, species richness , highest mode(s) for topographic position (Mode TP) , and aspect (Mode aspect) , and top three most abundant species by major species groups identified in cluster analysis of forested plots on the property of Ferrum College, Franklin County, VA . Means with the same letter are not significantly different at p .::_ 0.05 . LiTu = L iriodendron tulip1fera , OxAr = Oxydendrum arboreum , Ac Ru = Acer rub rum , PiSt = Pin us strobus, QuPr = Quercus prinus, AiAl = A ilanthus altissima , QuCu = Quercus coccinea . Species N DBH Density Species Mode Mode Most abundant Group (cm) (#Iha) Richness TP Aspect species 5 26 .2 a 540 a 5.4 a Toes lope NE LiTu, AcRu , OxAr 2 5 24.4 ab 495 a 6.0 a Side slope NE OxAr, AcRu , QuPr Shoulder SE 3 8 20 .2 b 756 b 5.1 a Ridgetop SW PiSt, QuCo, QuPr Shoulder most abundant species is summarized in Table 3. According to cluster analysis (Figure 1 ), the first cluster of plots 1, 11, 5, 3 and

New Records, Biogeography, and Habitat Protection Needs of Four Species of Potamon (Decapoda: Brachyura) in Greece

Abstract: Objectives are to determine the occurrence of species of Potamon in eastern Crete and the Aegean Islands (Chios, Naxos, Paros, Mykonos, Tinos and Andros); generate phylogenetic relationships among species to propose a biogeographic hypothesis relative to current distributions of the four species of the freshwater crab genus, Potamon, in Greece; and comment on the need to protect habitat suitable for the survival of species of Potamon in the country. Our collections, made in areas not previously sampled by researchers, indicate the presence of Potamon fluviatile on Tinos, N axos, and Andros, and Potamon potamios from central to eastern Crete; and verified the presence of Potamon ibericum on Chios. Cladistic analyses resulted in a single parsimonious tree (CI=85, RI=75). Potamon in the Balkan peninsula and islands in the Mediterranean region is a monophyletic group composed of two main clades: Clade 1 (P.fluviatile andPotamon algeriense) and Clade 2 (P. ibericum and its sister group composed of Potamon rhodium and Potamon potamios). Vicariant events (e.g. marine transgression and regression, orogeny, volcanism) are hypothesized as major factors that have shaped current distributions of species of Potamon in the Balkan Peninsula, Asia Minor, and the islands of the North Aegean Sea, Eastern Sporades, the Cyclades, and Crete. We recommend an increase in environmental education and communication among older and younger generations, agriculturalists, politicians, policy writers, land developers and economists to create an understanding for the need to protect land and aquatic environments that harbor unique species and the potential benefits for economic activities such as ecotourism. We also recommend the creation of an action plan to develop ecotourism around conservation areas ( e.g. from the source of existing springs downstream for about 200 m before the installation of water withdrawal equipment for irrigation and potable supplies) to generate revenue for funding protection initiatives and to promote green economic development that is ecologically and socio-culturally sustainable.

Induction of Winter Breeding in Small Mammals by 6-MBOA

Abstract: A plant-derived cyclic carbamate, 6-methoxybenzoxazolinone (6-MBOA), is known to initiate reproductive activity in Microtus montanus. We studied overwintering populations of four other rodent species, and observed increased reproduction in experimental populations of two herbivorous species, Microtus pennsylvanicus and Sigmodon hispidus, but not in two omnivorous species, Oryzomys palustris and Mus musculus. These results suggest that low concentrations of 6-MBOA may trigger seasonal breeding primarily in herbivorous small mammals. INTRODUCTION Many species of herbivorous mammals appear to initiate breeding after ingesting green vegetation ( e.g., Batzli and Pitelka 1971, Reynolds and Turkowski 1972, Van De Graaf and Balda 1973, Negus and Berger 1977). Later, Berger et al. (1981) demonstrated that reproduction in one small herbivore, Microtus montanus, the montane vole, is cued by 6-methoxybenzoxazolinone (6-MBOA) and/or its precursor 2, 4-Dihydroxy-7methoxy-2H-l,4-benzoxasin-3-(4H)-one (DIMBOA), which is common in seedlings and in rapidly growing plant parts (Sanders et al. 1981 ). Berger et al. (1981) suggested that 6-MBOA might elicit reproductive responses in many mammals because its site of action is high in the neuroendocrine system. We sought to examine the generality of the effects of 6-MBOA in initiating breeding among seasonally reproducing mammals by using a phylogenetically diverse group of four rodents studied during the winter months, the usual period of non-breeding for many rodents. Microtus pennsylvanicus (meadow vole) and Sigmodon hispidus (hispid cotton rat), which are almost exclusively herbivorous (Zimmerman 1965, Fleharty and Olson 1969), responded to the stimulation of 6-MBOA by increasing their levels of reproduction in midwinter, but the omnivorous Oryzomys palustris (marsh rice rat) and Mus mus cu/us (house mouse) did not respond (Negus et al. 1961, Rose and McGurk 2006, Miller and Webb 2001 ). STUDY AREA AND METHODS This experimental field study was conducted during the winter months of December to February. We set four 0.25-ha live-trapping grids at each of two sites in southeastern Virginia. At one site, with oldfields dominated by the perennial grass Fescue (Festuca spp. ), pairs of grids were separated by a highway interchange in the Bowers Hill section 144 VIRGINIA JOURNAL OF SCIENCE of Chesapeake, Virginia. The other study site was located in non-tidal marshes on Fisherman Island, a small barrier island 40 km north, at the entrance to the Chesapeake Bay, where American beach grass (Ammophila breviligulata) and wax myrtle (Myrica cerifera) were dominant. The tidal creek that separated the pairs of grids isolated at least the grid populations of house mice. At each location the Fitch live traps on two 5 by 5 grids, with 12.5-m intervals, received supplemental food while the other two grids received sham-treated food. (In all respects, the experimental protocol followed Berger et al. [ 1981].) The food was in the form of rolled oats bait coated with 6MBOA dissolved in ether and ethanol (4:1) at a concentration of 40 μg 6-MBOA g-1 oats (treatment) or solvent only (sham-treated control). Solvent was allowed to evaporate. The 6-MBOAor sham-treated oats were placed inside the locked-open traps (and replenished every third day) between periods of active trapping, making the traps into feeding stations. Trapping was conducted during weeks 1, 3, and 5. Each rodent was marked with a numbered ear tag, and externally evaluated for sex and reproductive condition before its release. Traps were locked open between trapping periods, permitting free access to the bait. After 5 weeks, animals were live trapped and removed to the laboratory for necropsy. To determine baseline levels of winter breeding activity, samples of rodents were collected in habitat contiguous with the trapping grids at the start of the study in late December. At the end of the experiment, animals were removed from the trapping grids, euthanized, and necropsied for signs of reproductive activity. Testes were weighed to the nearest mg, and the cauda epididymides were examined for the convolutions that indicate the presence of mature sperm (Jameson 1950). Uteri were examined for visible signs of pregnancy, ovaries for evidence of ovulation ( corpora lutea ), and reproductive tracts were weighed to the nearest mg. Animals were necropsied without knowledge of whether they had been removed from control or experimental grids until all necropsies had been completed. Then, an animal's treatment ( 6-MBOA or sham) was determined by examining its field tag number. Tests for differences (P < 0.05) in body and gonadal weights of pretrial, control, and treatment animals were made using one-way analysis of variance (ANOVA), and identification of differing group means was made by using Least Significance Difference tests. Student's tand Chi-square tests also were used. RESULTS Only Microtus showed evidence of reproduction in late December at the start of the study (Table 1 ), with one of eight females being pregnant and one-third of males judged to be fertile. The other three species showed the typical pattern of temperate rodents in the wintertime: no embryos or corpora lutea in females and regressed testes and looped epididymides in males. The 6-MBOA appeared to have had a variable effect on the four rodent species. Reproductive organ weights were significantly larger in both sexes of M pennsylvanicus and in male Sigmodon (Table 1 ). This pattern was consistent with other evidence of reproductive activity. For example, a significantly greater proportion of the adult female Microtus was pregnant on the experimental grids ( 10/22) than on the control grids (3/20, X = 4.55, P < 0.05) and the mean weights of litters were significantly heavier (4761.6 ± 1162.77 SE mg, treatment; 896.7 ± 564.07 SE mg, control; t = 2.92, P < 0.025), suggesting they had become pregnant earlier and were INDUCED WINTER BREEDING IN RODENTS ,:E ..... ·....... Q crj ;;>01-->-[~ ..... c "g"'O>->"" O!J ..... 1-< ~~~e.s~ 5'-"QO... en O 2 .s . t ~ II ~ 0 § c.)Q1-;1-<μ... Q -, c.8 8 ..... ·Q "'O ...i::: 0.. .s O "@ ~ -~ Cl) en ..0 .,... Q ;:l O!l E ·>-, ....... < ·v 4-;"'0~84-;;;> :~~~o~ u ·.,... ;:l en -, ~O!J

The Effects of Thawing Procedure and Supplementation on the Motility and Viability of Frozen-thawed Boar Semen

Abstract: The effect of two thawing procedures on frozen boar semen and supplementations to the fertilization media were studied. Frozen boar semen was thawed using either Percoll gradient or phosphate buffered saline (PBS) procedure . Supplementations were 1.0 mM L-glutamate, 1.0 mM N-acetylcysteine (NAC) , and 1.0 mM NAC-amide (NACA). Spermatozoa were analyzed for forward progressive motility (FPM) and viability every 0.5 h for 3 .0 h post-thawing. There were significantly (P < 0.05) higher numbers of viable (76.0 ± 5.1 %) and FPM (30 .0 ± 2.4%) spermatozoa at 3.0 h postthawing using the PBS procedure compared to the Percoll gradient thawed spermatozoa (65.0 ± 3.9%; 10.0 ± 4.5 %, respectively). Supplementation of 1.0 mM L-glutamate, 1.0 mM NAC, or 1.0 mM NACA had no significant effect on spermatozoa viability regardless of the time post-thaw. Supplementation of 1.0 mM L-glutamate, 1.0 mM NAC, or 1.0 mM NACA had no significant effect on FPM up to 1.0 h post-thaw. Spermatozoa with no supplementation or 1.0 mM L-glutamate had significantly higher (P < 0.05) FPM compared to the 1.0 mM NAC and 1.0 mM NACA supplemented groups at 1.5, 2 .0, 2 .5, and 3.0 h post-thaw. There was no significant difference between no supplementation or 1.0 mM L-glutamate on FPM regardless of the time post-thaw. There was no significant difference between 1.0 mM NAC or 1.0 mM NACA on FPM regardless of the time post-thaw. These results indicate that thawing procedure has an effect on spermatozoa viability and FPM but supplementation does not have an effect on the overall viability of spermatozoa during thawing, but may reduce FPM. INTRODUCTION Swine are valuable to science because they serve as a comparable model for human anatomy and physiology research. Research using swine as the experimental model often utilizes in vitro techniques to limit the experimental variability observed in vivo. The in vitro production of embryos is not as efficient as its natural counterpart, as a result of a decrease in the fertilization success rate during in vitro fertilization (IVF) and a reduction in viable !VF-derived embryos (Abeydeera 2002). 1 Corresponding Author: B. D. Whitaker, Ferrum College , 227 Garber Hall, 80 Wiley Drive, Ferrum VA, 24088, USA. bwhitaker@ferrum.edu . (540)365-4363 186 VIRGINIA JOURNAL OF SCIENCE Inefficiencies associated with in vitro production can be divided into two categories, the technique used to thaw frozen semen (Henkel et al. 2003, Chou et al. 2005) and the resulting media in which the semen is extended and cultured (Armstrong et al. 1999). Semen can be thawed using a saline wash procedure (Whitaker and Knight 2004, Chou et al. 2005) or Percoll gradient separation (Henkel et al. 2003 ). Both methods have variable results between replications and neither is close to the results obtained from fresh semen (Henkel et al. 2003 ). Current speculation is that oxidative stress during the freezing and thawing of semen produces large amounts of reactive oxygen species (ROS) that effects spermatozoa motility (Armstrong et al. 1999) and nuclear DNA (Fraser and Strzezek 2005). Antioxidants supplemented to the semen extender have been shown to improve the viability of the spermatozoa (Funahashi et al. 2005) and perhaps lessen the harmful effects of the free radicals on the spermatozoa (Blount et al. 2001 ). One antioxidant of interest is N-acetyl-cysteine (N AC) because it reduces cystine to cysteine, thus modulating glutathione (GSH) biosynthesis (Nakata et al. 1996, Issels et al. 1998); and has the ability to supply its sulfbydryl group to enhance glutathione-S-transferase activity (Nakata et al. 1996), which attaches glutathione to various compounds. Nacetyl-cysteine has been implicated in protecting against oxidative stress as seen when supplemented during the later stages of embryo development by decreasing the incidence of early fetal death (Xu et al. 2005). A derivative of NAC, NAC-amide (N ACA) is of interest because the amide addition neutralizes the otherwise negatively charged carboxyl group, allowing it to permeate most physiological membranes. Research has shown that NACA is able to replenish intracellular GSH and defend cells against oxidation (Grinberg et al. 2005, Wu et al. 2006). Therefore, the objectives of this study were to, 1) determine the effects of two different thawing procedures on spermatozoa viability and forward progressive motility (FPM), and 2) determine the effects of 1.0 mM L-glutamate, NAC, and NACA supplementation to the thawing media on spermatozoa viability and FPM. MATERIALS AND METHODS Media Unless otherwise stated, all chemicals were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). The Modena medium (152.6 mM glucose, 23.46 mM Na-citrate, 11.9 mM Na-carbonate, 6.99 mM EDTA, 26.66 mM Tris, and 15.1 mM citric acid) was adjusted to pH 7 .3. The PBS thawing medium was Dulbecco 's phosphate buffered saline (PBS) (Invitrogen, Carlsbad, CA, USA) containing 75 μg/mL potassium penicillin, 50 μg/mL streptomycin sulfate, and 0.1 % BSA (fraction V; 43H 1097, initial fraction by heat shock). The fertilization medium was a modified Tris-buffered medium (mTBM) formulated by Abeydeera and Day (1997). The N ACA was supplied by N ovia Pharmaceuticals Ltd. (Haifa Bay, Israel). Semen Frozen semen was obtained from Swine Genetics International Ltd. (Cambridge, IA, USA). The semen was extended using a yolk-based media and frozen in 5.0 mL straws in a Cryo-Med chamber to control the freezing rate. Upon arrival in the laboratory, straws were cut into 1.0 cm long pellets and stored in liquid nitrogen until use. All semen used was from the same boar but not from a single collection. EFFECTS ON THAWING OF BOAR SEMEN 187 Semen thawing using Percoll gradient A frozen semen pellet was thawed in 2.0 mL Modena and then overlain on a _Percoll gradient (2.0 mL 90% Percoll medium (v:v, Modena) overlain with 2.0 mL 45% Percoll medium (v:v, Modena)). The gradient was centrifuged at 700 x g for 20 min. The semen was then washed by centrifugation at 500 x g for 5 min in mTBM. After washing, the spermatozoa pellet was re-suspended with mTBM to a concentration of 4 x 10 spermatozoa/mL and incubated at 39°C in 5% CO 2 until analysis. Semen thawing using PBS wash A frozen semen pellet was thawed as previously described (Whitaker and Knight 2004). Briefly, the semen pellet was thawed in PBS at 39°C and centrifuged at 36.3 x g for 5 min. The semen was then washed by centrifugation twice at 553 x g for 5 min. After washing, the spermatozoa pellet was re-suspended with mTBM to a concentration of 4 x 10 spermatozoa/mL and incubated at 3 9°C in 5 % CO 2 until analysis. Viability staining Membrane integrity was assessed by staining spermatozoa with Eosin red and Aniline blue dye and then smeared on a microscope slide to determine viability. Spermatozoa that had intact membranes did not incorporate the dye and stained pink, whereas spermatozoa that had degraded membranes incorporated the dye and stained purple (Colenbrander et al. 2002). A total of 100 cells were counted for each pellet analyzed using a phase-contrast microscope at 400X magnification. Motility Forward progressive motility was analyzed by placing 20 μL of spermatozoa in 0.1 M sodium citrate buffer (v:v) on a 3 8°C glass slide. A total of 100 cells were either classified as either FPM or non-FPM for each pellet analyzed using a phase-contrast microscope at 400X magnification. Experiment 1: Comparison of semen thawing methods on spermatozoa viability and FPM A frozen semen pellet was thawed using either the PBS wash or the Percoll gradient procedure and then analyzed for viability and FPM at 3 .0 h post-thawing. For this experiment a total of 60 semen pellets were used, by thawing and analyzing 30 pellets for each procedure. Experiment 2: Comparison of fertilization media supplements on spermatozoa viability and FPM Based on the results from Experiment 1, a frozen semen pellet was thawed using the PBS wash procedure and incubated in mTBM supplemented with either 1.0 mM L-glutamate, N AC, or N ACA for 3 .0 h. Spermatozoa were analyzed for viability and FPM every 0.5 h for 3.0 h post-thaw. For this experiment a total of 90 semen pellets used, by thawing and analyzing 30 pellets for each supplement. Statistical Analysis Data in both experiments were analyzed by one-way ANOV A using the PROC ANOV A procedures of SAS (SAS Institute, Cary, NC) because the data were balanced in all cases. When there was a significant effect of treatment, significant differences were determined using LSMEANS statement and Tukey adjustment for multiple comparisons. The effects included in the model were treatment and technician. Technician effects were not significant (P < 0.05) and deleted from the final model. A probability ofless than 0.05 (P < 0.05) was considered significant. 188 VIRGINIA JOURNAL OF SCIENCE a PBS Wash 100 • Percell Gradient 90 a 80 (t'l 0 70 N 0 ..... co 60 E s... 8. 50 en