Timothy S. Gross

Embryonic development in superovulated dairy cattle exposed to elevated ambient temperatures between the onset of estrus and insemination

Holstein heifers (n = 16) were used to determine whether heat stress prior to ovulation increases the incidence of embryonic abnormalities. Heifers were superovulated with Follicle Stimulating Hormone (FSH-P; 32 mg total), beginning on Days 10 or 11 of the estrous cycle. Prostaglandin F2α (Lutalyse; 60 mg total) was administered on Day 3 of FSH-P treatment. Heifers were maintained at either thermoneutrality (24°C) or under hyperthermic conditions (exposure to 42°C for 10 h) beginning at the onset of estrus. Following artificial inseminations at 15 and 20 h after the onset of estrus, heifers were continuously maintained under environmental conditions of thermoneutrality for 7 days as provided by environmental shade structures. On Day 7 post estrus, embryos were recovered nonsurgically and evaluated morphologically for stage of development and quality. The distribution of embryos classified as normal, retarded and/or abnormal, or as unfertilized ova differed (P < 0.001) between heat stress and thermoneutral treatments. Only 12.0% of 25 embryos recovered from heat-stressed heifers were normal compared with 68.4% of 19 embryos from thermoneutral heifers. Stressed heifers had a higher (P < 0.001) incidence of retarded and/or abnormal embryos with degenerated blastomeres. These data indicate that thermal stress during the periovulatory period increases the incidence of retarded and/or abnormal embryos in superovulated heifers.

Environmental enrichment of brown capuchins (Cebus apella): Behavioral and plasma and fecal cortisol measures of effectiveness

No consensus exists about the quantity and variety of environmental enrichment needed to achieve an acceptable level of psychological well‐being among singly housed primates. Behavioral and plasma and fecal cortisol measures were used to evaluate the effectiveness of four levels of toy and foraging enrichment provided to eight wild‐caught, singly housed adult male brown capuchins (Cebus apella). The 16‐week‐long study comprised six conditions and began with a 4‐week‐long preexperimental and ended with a 4‐week‐long postexperimental period during which the subjects were maintained at baseline enrichment levels. During the intervening 8 weeks, the subjects were randomly assigned to a sequence of four 2‐week‐long experimental conditions: control (baseline conditions), toy (the addition of two plastic toys to each cage), box (access to a foraging box with food treats hidden within crushed alfalfa), and box & toy (the addition of two plastic toys and access to a foraging box). Behavioral responses to changes in enrichment were rapid and extensive. Within‐subject repeated‐measure ANOVAs with planned post hoc contrasts identified highly significant reductions in abnormal and undesirable behaviors (and increases in normal behaviors) as the level of enrichment increased from control to toy to box to box & toy. No significant behavioral differences were found between the control and pre‐ and postexperimental conditions. Plasma and fecal cortisol measures revealed a different response to changing enrichment levels. Repeated‐measure ANOVA models found significant changes in both these measures across the six conditions. The planned post hoc analyses, however, while finding dramatic increases in cortisol titers in both the pre‐ and postexperimental conditions relative to the control condition, did not distinguish cortisol responses among the four enrichment levels. Linear regressions among weekly group means in behavioral and cortisol measures (n = 16) found that plasma cortisol was significantly predicted by the proportions of both normal and abnormal behaviors; as the proportion of normal behaviors increased, the plasma cortisol measures decreased. Plasma cortisol weekly group means were also significantly and positively predicted by fecal cortisol weekly group means, but no behavioral measure significantly predicted fecal cortisol weekly group means. In sum, these findings argue strongly that access to a variety of toy and foraging enrichment positively affects behavioral and physiological responses to stress and enhances psychological well‐being in singly housed brown capuchins. Am. J. Primatol. 48:49–68, 1999.

An evaluation of biomarkers of reproductive function and potential contaminant effects in Florida largemouth bass (Micropterus salmoides floridanus) sampled from the St. Johns River

The objective of this study was to describe and compare several reproductive parameters for Florida largemouth bass (Micropterus salmoides floridanus) inhabiting the St. Johns River and exposed to different types and/or degrees of contamination. Welaka was selected as the reference site in this study because of its low urban and agricultural development, Palatka is in close proximity to a paper mill plant, the Green Cove site is influenced by marine shipping activities and Julington Creek site receives discharges of domestic wastewater and storm water runoff from recreational boating marinas. For this study, bass were sampled both prior to (September 1996) and during the spawning season (February 1997). In order to characterize chemical exposure, bass livers were analyzed for up to 90 trace organics and 11 trace metal contaminants. Reproductive parameters measured included gonadosomatic index (GSI), histological evaluation of gonads and plasma concentrations of vitellogenin (VTG), 17beta-estradiol (E2) and 11-ketotestosterone (11-KT). In general, the sum of organic chemicals was highest in livers from Palatka bass and bass from Green Cove and Julington Creek had higher hepatic concentrations of low molecular polycyclic aromatic hydrocarbons and polychlorinated biphenyls when compared to fish from Welaka. Metals were more variable across sites, with highest mean concentrations found in bass from either Julington Creek (Ag, As, Cr, Cu, Zn) or Welaka (Cd, Hg, Pb, Se, Tn). Female bass from Palatka and Green Cove had lower concentrations of E2, VTG and lower GSI in relation to Welaka. Males from Palatka and Green Cove showed comparable declines in 11-KT in relation to males from Julington Creek and GSI were decreased only in Palatka males. These results indicate a geographical trend in reproductive effects, with changes being most pronounced at the site closest to the paper mill (Palatka) and decreasing as the St. Johns River flows downstream. Since reproductive alterations were most evident in bass sampled from the site closest to the paper mill discharge, it is possible that exposure to these effluents might explain at least some of the results reported here. However, the presence of reproductive alterations in fish sampled at a considerable distance from the mill discharge (Green Cove, 40 km) would suggest exposure to chemicals released from sources other than the paper mill plant. It is clear that additional studies are needed to evaluate the potential impact of these reproductive changes in populations of Florida largemouth bass inhabiting the St. Johns River.

Indoor husbandry of the panther chameleon Chamaeleo [Furcifer] pardalis: Effects of dietary vitamins A and D and ultraviolet irradiation on pathology and life-history traits

To assess the importance of diet and light for indoor maintenance, hatchling panther chameleons were reared for 1 year on crickets fed diets that differed in vitamin concentrations and in different light environments. Dietary transfer of vitamins from the cricket diet to the lizards via the crickets was quantified, as was UV irradiance. There was a statistically significant dietary enhancement of growth by both vitamins on males. UV-A irradiation significantly suppressed growth of females. Low vitamin A shortened life span and resulted in a number of gross and histological pathologies. Hepatocellular lipidosis, indicating a possible toxicosis, occurred with all diets and light treatments. Higher vitamin A resulted in mild soft-tissue mineralization, and high vitamin D shortened the life span of females. Low vitamin A drastically reduced reproduction in both sexes. The intermediate levels of dietary vitamins resulted in the best production of viable eggs by females. However, without high UV-B irradiation, all viable eggs died at term and contained different vitamin levels than hatching eggs from wild-caught females. Baseline levels of egg calcium are given for hatching eggs from wild-caught females. Modifications in current husbandry procedures are recommended.

Presence of an intracellular endometrial inhibitor of prostaglandin synthesis duing early pregnancy in the cow

Previous studies have detected reduced endometrial secretion of prostaglandins during pregnancy in cattle. The present experiment tested the hypothesis that reduced secretion of prostaglandins is caused by induction of an intracellular endometrial inhibitor of prostaglandin synthesis. The microsomal fraction of parturient bovine cotyledons was utilized as a source of enzymes for prostaglandin synthesis. Endometrial tissues collected at Day 17 of the estrous cycle (n = 12) and pregnancy (n = 12) were homogenized and subjected to differential centrifugation for preparation of microsomes and a high-speed (100,000 x g) cytosolic supernatant. Endometrial intracellular preparations were then examined for the ability to modulate prostaglandin synthesis by cotyledonary microsomes from parturient cows. Endometrial intracellular preparations from cyclic cows decreased (P less than 0.05) PGF synthesis by cotyledonary microsomes to a slight extent (supernatant, 21% reduction; microsomes, 11% reduction), while preparations from pregnant cows markedly decreased (P less than 0.01) PGF synthesis (supernatant, 63% reduction; microsomes, 28% reduction; supernatants vs microsomes, P less than 0.01). Regardless of the amount of arachidonic acid available as substrate (25-400 micrograms) endometrial supernatant from pregnant cows (pooled sample) caused a 50% inhibition (IC50) of prostaglandin synthesis at a tissue equivalent of 270 +/- 9.1 mg. The mechanism of inhibition by endometrial high-speed supernatant from pregnant cows appears to be non-competitive with respect to arachidonic acid. The inhibitor(s) may be proteinaceous (70-75 kDa and 25-35 kDa) and can be precipitated by 20% saturated ammonium sulfate. In conclusion, early pregnancy in cattle appears to be associated with increased amounts of an intracellular endometrial inhibitor of prostaglandin synthesis.

Decreased glutathione S-transferase expression and activity and altered sex steroids in Lake Apopka brown bullheads (Ameiurus nebulosus).

A number of freshwater lakes and reclaimed agricultural sites in Central Florida have been the receiving waters for agrochemical and municipal runoff. One of these sites, Lake Apopka, is also a eutrophic system that has been the focus of several case studies reporting altered reproductive activity linked to bioaccumulation of persistent organochlorine chemicals in aquatic species. The present study was initiated to determine if brown bullheads (Ameiurus nebulosus) from the north marsh of Lake Apopka (Lake Apopka Marsh) exhibit an altered capacity to detoxify environmental chemicals through hepatic glutathione S-transferase (GST)-mediated conjugation as compared with bullheads from a nearby reference site (Lake Woodruff). We also compared plasma sex hormone concentrations (testosterone, 17-beta estradiol, and 11 keto-testosterone) in bullheads from the two sites. Female bullheads from Lake Apopka had 40% lower initial rate GST conjugative activity toward 1-chloro-2,4-dinitrobenzene (CDNB), 50% lower activity towards p-nitrobutyl chloride (NBC), 33% lower activity toward ethacrynic acid (ECA), and 43% lower activity toward Delta5-androstene-3,17-dione (Delta(5)-ADI), as compared with female bullheads from Lake Woodruff. Enzyme kinetic analyses demonstrated that female bullheads from Lake Apopka had lower GST-catalyzed CDNB clearance than did female Lake Woodruff bullheads. Western blotting studies of bullhead liver cytosolic proteins demonstrated that the reduced GST catalytic activities in female Lake Apopka bullheads were accompanied by lower expression of hepatic GST protein. No site differences were observed with respect to GST activities or GST protein expression in male bullheads. Female Lake Apopka bullheads also had elevated concentrations of plasma androgens (testosterone and 11-ketotestosterone) as compared with females from Lake Woodruff. In contrast, male Lake Apopka bullheads had elevated levels of plasma estrogen but similar levels of androgens as compared with male bullheads from Lake Woodruff. Collectively, our studies indicate the presence of reduced GST protein expression, reduced GST conjugative capacity and altered sex steroid homeostasis in female bullheads from a contaminated field site in Central Florida. The implications of these physiological alterations in terms of pollutant biotransformation and reproduction are discussed.

Morphometric and Histopathological Parameters of Gonadal Development in Adult Common Carp from Contaminated and Reference Sites in Lake Mead, Nevada

Abstract This study examined the hypothesis that exposure to sublethal concentrations of contaminants alters the gonadal condition of feral common carp Cyprinus carpio. Adult common carp in Lake Mead, Nevada, were collected from a contaminated site (Las Vegas Bay) that receives municipal and industrial effluent and from a reference site (Overton Arm) with a relatively low level of contamination. Fish were sampled seven times over a 1-year period extending over two separate spawning seasons. Morphometric and histopathological parameters of gonadal and germ cell development were determined. In males, the pattern of seasonal changes in the gonadosomatic index (GSI) was similar between the sites and showed no clear association with site-specific seasonal temperature profiles. However, Las Vegas Bay males had consistently lower GSI values and, on one of the sampling dates, a lower proportion of sperm relative to other germ cell stages (determined histologically). Further, Las Vegas Bay males had a higher incid...

Prostaglandin secretion by perifused porcine endometrium: further evidence for an endocrine versus exocrine secretion of prostaglandins

Bilateral perifusion devices were utilized for measurement of prostaglandin secretion by luminal and myometrial surfaces of porcine endometrium. Tissues were collected from Days 10, 12 and 14 pregnant, Day 14 cyclic and Day 14 estrogen-induced pseudopregnant gilts. Each tissue was placed into duplicate perifusion devices and perifused with Krebs-Ringer Bicarbonate solution at 3 ml/10 min for 2 h, fractions collected every 10 min and oxytocin (1 IU/ml) perifused during fractions 6-10 to the luminal side of one chamber and to the myometrial side of the other chamber. Secretion rates of PGF were higher (P less than 0.05) than PGE2 for each status. Secretion rates of PGF and PGE2 were higher (P less than 0.01) from the luminal side for Day 12 pregnant, Day 14 pregnant and Day 14 pseudo-pregnant gilts, whereas secretion was higher from the myometrial side for Day 10 pregnant and Day 14 cyclic gilts. Oxytocin increased (P less than 0.01) prostaglandin secretion from the luminal side regardless of reproductive status. Pregnancy at Day 12 and Day 14, as well as estrogen treatment, were associated with prostaglandin secretion in a luminal (exocrine) orientation versus a myometrial (endocrine) orientation for Day 14 cyclic and Day 10 pregnant gilts. These data indicate an estrogen associated switch between Days 10 and 12 of pregnancy from an endocrine to an exocrine secretion of prostaglandins.

Terrestrial predator alarm vocalizations are a valid monitor of stress in captive brown capuchins (Cebus apella)

The vocal behavior of captive animals is increasingly exploited as an index of well-being. Here we show that the terrestrial predator alarm (TPA) vocalization, a robust and acoustically distinctive anti-predation vocal response present in many mammal and bird species, offers useful information on the relative well-being and stress levels of captive animals. In a 16-week experiment evaluating the effects of varying levels of physical environmental enrichment (control < toys < foraging box < foraging box and toys) in the cages of eight singly housed adult male brown capuchins, we quantified the 1) emission rate of TPAs, 2) proportions of normal and abnormal behavior sample intervals, and 3) fecal and plasma cortisol levels. Variation in TPA emission across the experimental conditions was significant. We found significant reductions in the mean TPA production rate by the group in the enriched (toys, foraging box, and foraging box and toys) compared to the control condition; pre- and post-experimental conditions, however, did not differ from the control condition. Mean TPA production by the group was also significantly positively correlated to mean group levels of fecal cortisol and proportion of abnormal behavior sample intervals, and significantly negatively correlated to the average proportion of normal behavior sample intervals in the group. Based on group means, plasma cortisol levels were positively, but not significantly, related to increasing TPA rate. At the level of the responses of an individual subject, however, the covariation between the vocal and non-vocal behavioral measures and the cortisol assays seldom attained significance. Nevertheless, the direction of the relationships among these parameters within individual subjects typically mirrored those correlations based on group means. At both the group mean and individual levels, our results are consistent with the interpretation that in conditions of low environmental enrichment the study subjects were more stressed, and therefore more reactive to the presence of a threatening terrestrial stimulus (human observer), than when in more enriched conditions. We suggest that protocols to evaluate the effectiveness of enrichment for captive species other than brown capuchins could also profitably exploit TPAs as a first-line monitor or as corroboratory evidence of current well-being. Zoo Biol 18:295–312, 1999.

Predicting maternal body burdens of organochlorine pesticides from eggs and evidence of maternal transfer in Alligator mississippiensis

Few data exist regarding maternal-embryonal transfer of organochlorine pesticides (OCPs) in reptiles. The objective of the present study was to evaluate maternal transfer of OCPs in American alligators (Alligator mississippiensis) from low-, intermediate-, and high-OCP-exposure sites. Overall, total OCP burdens ranged from less than 0.8 ppb in blood to more than 44,000 ppb in abdominal adipose tissue (wet wt concentrations). Lipid-adjusted ratios of maternal adipose burdens (total OCPs) to yolk burdens were close to one (0.94 +/- 0.31:1), suggesting that animals were in steady state and that OCPs in eggs originated from adipose lipids. In contrast, lipid-adjusted muscle and liver OCP burdens were greater than yolk OCP burdens, suggesting that lipids in muscle were not utilized during oogenesis and that nonlipid liver tissue sequesters OCPs. Predictive equations were derived for several tissues and several OCP analytes with r2 values ranging from 0.40 to 0.99 (p < 0.05). We suggest that yolk burdens are predictive of maternal tissue burdens for certain tissues and OCPs and that certain OCPs are maternally transferred in the American alligator. Furthermore, we suggest that future studies should investigate the applicability of these predictive equations for assessing maternal exposure in other crocodilian species.