Superovulation of Beef Heifers with Follicle Stimulating Hormone or Human Menopausal Gonadotropin: Acute Effects on Hormone Secretion

The effects of superovulatory treatment (follicle stimulating hormone [FSH] versus human menopausal gonadotropin [HMG]) and of route of administration (intramuscular versus intravenous) of prostaglandin F2a (PGF2a) on hormonal profiles were determined in 32 Angus x Hereford heifers for breeding and subsequent embryo collection and transfer. Heifers were superstimulated either with FSH (total of 26 milligrams) or HMG (total of 1,050 international units) beginning on days 9 to 12 of an estrous cycle and PGF2a (40 milligrams) was administered at 60 and 72 hours after the beginning of superovulatory treatments. Heifers were artificially inseminated three times at 12-hour intervals beginning 48 hours after PGF2a treatment. Blood serum samples were collected immediately before treatments began and at frequent intervals until embryo collection 288 hours later. Concentrations of luteinizing hormone (LH) and FSH were not affected by hormone treatments, route of PGF2a injection, or interactions between them. Estradiol-17ß (E2-17ß) levels were higher in HMG- than in FSH-treated heifers 60 hours after gonadotropin treatment. Peak concentration of E2-17ß occurred earlier in HMGthan in FSH-treated heifers and earlier in heifers injected with PGF2a intramuscularly than those injected intravenously. Progesterone concentrations were not influenced by treatment or route of PGF2a administration. The progesterone:E2-17ß ratio was higher in FSH- than in HMG-treated heifers 24 hours after the LH peak. The high steroid hormone concentrations in superovulated beef heifers before and after ovulation may lead to asynchrony between stages of embryonic development, a situation that may interfere with the pregnancy outcome of superovulated embryos in recipient animals.

Aromatase deficiency caused by a novel P450arom gene mutation: impact of absent estrogen production on serum gonadotropin concentration in a boy.

We identified a new point mutation in the CYP19 gene responsible for aromatase (P450arom) deficiency in a 46,XY male infant with unremarkable clinical findings at birth. This boy is homozygote for a 1-bp (C) deletion in exon 5 of the aromatase gene causing a frame-shift mutation. The frame-shift results in a prematurely terminated protein that is inactive due to the absence of the functional regions of the enzyme. Aromatase deficiency was suspected prenatally because of the severe virilization of the mother during the early pregnancy, and the diagnosis was confirmed shortly after birth. Four weeks after birth, the baby boy showed extremely low levels of serum estrogens, but had a normal level of serum free testosterone; in comparison with the high serum concentration of androstenedione at birth, a striking decrease occurred by 4 weeks postnatally. We previously reported elevated basal and stimulated FSH levels in a female infant with aromatase deficiency in the first year of life. In contrast, in the male infant, basal FSH and peak FSH levels after standard GnRH stimulation tests were normal. This finding suggests that the contribution of estrogen to the hypothalamic-pituitary gonadotropin-gonadal feedback mechanism is different in boys and girls during infancy and early childhood. In normal girls, serum estradiol concentrations strongly correlate with circulating inhibin levels, and thus, low inhibin levels may contribute to the striking elevation of FSH in young girls with aromatase deficiency. In contrast, estradiol levels are physiologically about a 7-fold lower in boys than in girls, and serum inhibin levels remain elevated even though levels of FSH, LH, and testosterone are decreased.

Use of Electroshock for Euthanizing and Immobilizing Adult Spring Chinook Salmon in a Hatchery

This study evaluated the use of electroshock as in alternative to traditional techniques for immobilizing and euthanizing hatchery fish. We used a commercially available electroanesthesia unit at the U.S. Fish and Wildlife Service's Carson National Fish Hatchery (Carson, Washington) to euthanize adult spring Chinook salmon Oncorhynchus tshawytscha and to son and collect gametes of fish at maturation. During euthanization by electroshock, the response of each fish was observed, Muscular and vertebral hemorrhaging wits quantified, and electrical settings were optimized accordingly. During gamete collection, fish were either electroshocked or exposed to tricaine methanesulfortate (MS-222); hemorrhaging, egg viability. egg size and quantity, and resultant fry quality were examined for each treatment group. Electroshocked fish had a higher likelihood Of injury during gamete collection than did fish exposed to MS-222. On average, each electroshocked fish had less than two hemorrhages oil both fillets examined. The size of each hemorrhage was less than 0.10% of the fillet surface. Fecundity and egg and fry quality were not affected by either immobilization method. Electroshock was a viable and efficient means of euthanizing adult spring Chinook salmon or sorting the fish and collecting their gametes. However, equipment settings must be optimized based on site-specific (e.g., water conductivity) and species-specific (e.g., fish size and seasonal state of maturation) factors.

The Effects of Smolt Stocking Strategies on Migratory Path Selection of Adult Atlantic Salmon in the Penobscot River, Maine

Understanding the homing behavior of Atlantic salmon Salmo salar is vital to the restoration program employed on the Penobscot River, Maine. To produce significant adult returns, managers currently stock hatchery-raised smolts in specific river sections, providing smolts the opportunity to imprint on chemical signals and enabling their return to productive spawning and rearing habitat as adults. In this study, we used observational evidence from passive integrated transponder telemetry to determine whether adults returning from smolt stockings behaved in a way that suggested strong homing to smolt stocking locations. Adults returning from smolt stocking locations located in or at the mouth of the Piscataquis River were more likely to be detected as entering the Piscataquis River than were adults returning from the upper Penobscot River smolt stocking locations. In general, returning adult Atlantic salmon that had been stocked near or in tributaries as smolts chose a path more quickly than those that had been stocked in more downstream or main-stem locations. These results suggest that Atlantic salmon smolts should be stocked at specific sites with superior habitat for spawning kind juvenile survival to capitalize on the strong homing tendency in adults. This technique call also be utilized to allow for natural selection and the development of localized stocks.

Responses of Atlantic Salmon Parr to Output of Pulsed Ultrasonic Transmitters

The output from some pulsed ultrasonic transmitters commonly used in fish movement studies is faintly audible to humans. This study was undertaken to determine if the output from these and some other transmitters is detectable by Atlantic salmon (Salmo salar) parr. Classical conditioning of cardiac deceleration was attempted using the transmitter's output as the conditioned stimulus. The results from 29 experimental and 14 control fish suggest that the parr were unable to detect the output from these transmitters.

Descaling Injury Impairs the Osmoregulatory Ability of Atlantic Salmon Smolts Entering Seawater

The effect of descaling injury on the osmoregulatory ability of hatchery Atlantic salmon Salmo salar smolts in seawater was investigated. Experimental series were initiated during early, middle, and late periods of the spring smolt migration (April 25, May 11, and May 31, respectively). For each time series, descaled smolts (subjected to descaling on 10% of the body surface area) and control smolts (held out of water for 15 s) were transferred to seawater at 0, 1, 3, or 7 d posttreatment. After fish were held in 35% seawater for 24 h, gill and blood samples were collected and analyzed for Na(+),K(+)-ATPase activity and plasma osmolyte levels. Based on gill Na(+),K(+)-ATPase activity, the three series spanned the period from early smolting (increasing activity) to de-smolting (decreasing activity). In each series, descaled fish transferred to seawater at 0 and 1 d posttreatment had greater plasma osmolality than control fish; descaled fish transferred to seawater at 3 d posttreatment did not differ from controls. The greatest perturbation in osmolality (70 milliosmoles) was observed at the peak of smolting (middle series), whereas lesser increases were seen for early and late-series smolts. The observed osmotic perturbations in descaled fish would probably reduce performance and decrease survival during smolt migration.

Migratory Urge and Gill Na(+),K(+)-ATPase Activity of Hatchery-Reared Atlantic Salmon Smolts from the Dennys and Penobscot River Stocks, Maine

Hatchery-reared Atlantic salmon Salmo salar smolts produced from captive-reared Dennys River and sea-run Penobscot River broodstock are released into their source rivers in Maine. The adult return rate of Dennys smolts is comparatively low, and disparity in smolt quality between stocks resulting from genetic or broodstock rearing effects is plausible. Smolt behavior and physiology were assessed during sequential 14-d trials conducted in seminatural annular tanks with circular flow. "Migratory urge'' (downstream movement) was monitored remotely using passive integrated transponder tags, and gill Na(+),K(+)-ATPase activity was measured at the beginning and end of the trials to provide an index of smolt development. The migratory urge of both stocks was low in early April, increased 20-fold through late May, and declined by the end of June. The frequency and seasonal distribution of downstream movement were independent of stock. In March and April, initial gill Na(+),K(+)-ATPase activities of Penobscot River smolts were lower than those of Dennys River smolts. For these trials, however, Penobscot River smolts increased enzyme activity after exposure to the tank, whereas Dennys River smolts did not, resulting in similar activities between stocks at the end of all trials. There was no clear relationship between migratory urge and gill Na(+),K(+)-ATPase activity. Gill Na(+),K(+)-ATPase activity of both stocks increased in advance of migratory urge and then declined while migratory urge was increasing. Maximum movement was observed from 2 h after sunset through 1 h after sunrise but varied seasonally. Dennys River smolts were slightly more nocturnal than Penobscot River smolts. These data suggest that Dennys and Penobscot River stocks are not markedly different in either physiological or behavioral expression of smolting.

Acoustic Detection of Organic Enrichment in Sediments at a Salmon Farm Is Confirmed by Independent Groundtruthing Methods

Acoustic backscatter contrast in depositional sediments under salmon farm cages in the Bay of Fundy, Canada, was correlated with localized changes in (unknown) sediment geotechnical properties, as indicated by 4 independent measures of organic enrichment. Sediment total sulfides and redox potentials, enzyme hydrolyzable amino acids, sediment profile imaging and macrofaunal samples, taken at mid-cage positions, each rejected the null hypothesis that salmon cage footprints, defined acoustically as high backscatter areas, were indistinguishable from nearby reference areas. Acoustic backscatter imaging appears capable of mapping organic enrichment in depositional sediments caused by excessive inputs of salmon farm wastes associated with intensive aquaculture.

Immunization against gonadotropin-releasing hormone in dairy cattle: Antibody titers, ovarian function, hormonal levels, and reversibility

Suppression of cyclic activity in cattle is often desired in alpine farming and for feedlot cattle not intended for breeding. A cattle-specific anti-GnRH vaccination (Bopriva, Zoetis Australia Ltd., West Ryde, Australia) is approved for use in heifers and bulls in New Zealand, Australia, Mexico, Brazil, Argentina, Turkey, and Peru. Eleven healthy, cyclic Swiss Fleckvieh cows were included in the study and vaccinated twice with Bopriva 4wk apart. Injection site, rectal body temperature, and heart and respiratory rates were recorded before and 3d following each vaccination. Blood samples were taken weekly for progesterone and estrogen analysis and to determine GnRH antibody titer. Ovaries were examined weekly, using ultrasound to count the number of follicles and identify the presence of a corpus luteum. Thirty weeks after the first vaccination, the cows were subjected to a controlled internal drug-releasing device-based Select-Synch treatment. The GnRH antibody titers increased after the second vaccination and peaked 2wk later. Estrogen levels were not influenced by vaccination, and progesterone level decreased in 7 of 11 cows up to 3wk after the second vaccination and remained low for 10 to 15wk following the second vaccination. The number of class I follicles (diameter ≤5mm) was not influenced by vaccination, whereas the number of class II follicles (diameter 6-9mm) decreased between 7 and 16wk after the first vaccination. Class III follicles (diameter >9mm) were totally absent during this period in most cows. The median period until recurrence of class III follicles was 78d from the day of the second vaccination (95% confidence interval: 60-92d). After vaccination, all cows showed swelling and pain at the injection site, and these reactions subsided within 2wk. Body temperature and heart and respiratory rates increased after the first and second vaccinations and returned to normal values within 2d of each vaccination. The cows in our study were not observed to display estrus behavior until 30wk after the first vaccination. Therefore, a Select-Synch protocol was initiated at that time. Ten cows became pregnant after the first insemination (the remaining cow was reinseminated once until confirmed pregnancy). Bopriva induced a reliable and reversible suppression of reproductive cyclicity for more than 2mo. The best practical predictor for the length of the anestrus period was the absence of class III follicles.

Anti-Müllerian hormone and progesterone levels produced by granulosa cells are higher when derived from natural cycle IVF than from conventional gonadotropin-stimulated IVF.

BACKGROUND The study was designed to compare the effect of in vitro FSH stimulation on the hormone production and gene expression profile of granulosa cells (GCs) isolated from single naturally matured follicles obtained from natural cycle in vitro fertilization (NC-IVF) with granulosa cells obtained from conventional gonadotropin-stimulated IVF (c-IVF). METHODS Lutein granulosa cells from the dominant follicle were isolated and cultured in absence or presence of recombinant FSH. The cultures were run for 48 h and six days. Messenger RNA (mRNA) expressions of anti-Müllerian hormone (AMH) and FSH receptor were measured by quantitative polymerase chain reaction (qPCR). AMH protein and progesterone concentration (P4) in cultured supernatant were measured by ELISA and RIA. RESULTS Our results showed that the mRNA expression of AMH was significantly higher in GCs from NC- than from c-IVF on day 6 after treatment with FSH (1 IU/mL). The FSH stimulation increased the concentration of AMH in the culture supernatant of GCs from NC-IVF compared with cells from c-IVF. In the culture medium, the AMH level was correlated significantly and positively to progesterone concentration. CONCLUSIONS Differences in the levels of AMH and progesterone released into the medium by cultured GC as well as in AMH gene expression were observed between GCs obtained under natural and stimulated IVF protocols. The results suggest that artificial gonadotropin stimulation may have an effect on the intra-follicular metabolism. A significant positive correlation between AMH and progesterone may suggest progesterone as a factor influencing AMH secretion.

Rapid temperature-dependent wound closure following adipose fin clipping of Atlantic salmon Salmo salar L.

Three groups of Atlantic salmon were kept at a constant temperature of 4, 10 and 14 °C. The adipose fins were removed; six fish/group were sampled at 11 subsequent time points post-clipping. Samples were prepared for histopathological examination to study the course of re-epithelization. A score sheet was developed to assess the regeneration of epidermal and dermal cell types. Wounds were covered by a thin epidermal layer between 4 and 6 h post-clipping at 10 and 14 °C. In contrast, wound closure was completed between 6 and 12 h in fish held at a constant temperature of 4 °C. By 18 h post-clipping, superficial cells, cuboidal cells, prismatic basal cells and mucous cells were discernible in all temperature groups, rapidly progressing towards normal epidermal structure and thickness. Within the observation period, only minor regeneration was found in the dermal layers. A positive correlation between water temperature and healing rates was established for the epidermis. The rapid wound closure rate, epidermal normalization and the absence of inflammatory reaction signs suggest that adipose fin clipping under anaesthesia constitutes a minimally invasive method that may be used to mark large numbers of salmon presmolts without compromising fish welfare.

Effect of temperature and diet on wound healing in Atlantic salmon (Salmo salar L.).

Compromised skin integrity of farmed Atlantic salmon, commonly occurring under low temperature and stressful conditions, has major impacts on animal welfare and economic productivity. Even fish with minimal scale loss and minor wounds can suffer from secondary infections, causing downgrading and mortalities. Wound healing is a complex process, where water temperature and nutrition play key roles. In this study, Atlantic salmon (260 g) were held at different water temperatures (4 or 12 °C) and fed three different diets for 10 weeks, before artificial wounds were inflicted and the wound healing process monitored for 2 weeks. The fish were fed either a control diet, a diet supplemented with zinc (Zn) or a diet containing a combination of functional ingredients in addition to Zn. The effect of diet was assessed through subjective and quantitative skin histology and the transcription of skin-associated chemokines. Histology confirmed that wound healing was faster at 12 °C. The epidermis was more organised, and image analyses of digitised skin slides showed that fish fed diets with added Zn had a significantly larger area of the epidermis covered by mucous cells in the deeper layers after 2 weeks, representing more advanced healing progression. Constitutive levels of the newly described chemokines, herein named CK 11A, B and C, confirmed their preferential expression in skin compared to other tissues. Contrasting modulation profiles at 4 and 12 °C were seen for all three chemokines during the wound healing time course, while the Zn-supplemented diets significantly increased the expression of CK 11A and B during the first 24 h of the healing phase.

Prediction of Benthic Impact for Salmon Net-Pens Based on the Balance of Benthic Oxygen Supply and Demand

The ratio between oxygen supply and oxygen demand was examined as a predictor of benthic response to organic enrichment caused by salmon net-pen aquaculture. Oxygen supply to the benthos was calculated based on Fickian diffusion and near-bottom flow velocities. A strong linear correlation was found between measured carbon sedimentation rates and rates of benthic metabolism. This relationship allowed an estimation of oxygen demand based on sedimentation rates. Comparison of several production sites in Maine (USA) coastal waters showed that for sites where oxygen demand exceeded supply benthic impacts were high and for sites where oxygen supply exceeded demand benthic impacts were low. These findings were summarized in the form of a predictive model that should be useful in siting salmon production facilities.

Phthalate exposure among US women, NHANES 1999--2002: Relationship to serum gonadotropin levels

Phthalates are industrial chemicals used primarily as plasticizers though they and are found in a myriad of consumer goods such as children's toys, food packaging, dental sealants, cosmetics, pharmaceuticals, perfumes, and building materials. US biomonitoring data show more than 75% of the population have exposure to mono-n-butyl phthalate (MBP), mono-ethyl phthalate (MEP), mono-(2-ethyl) hexyl phthalate (MEHP), and mono-benzyl phthalate (MBZP). Reproductive toxicity from phthalate exposure in animal models has raised concerns about similar effects on fertility in humans. This dissertation research focuses on phthalate exposures in the US population and investigates the plausibility of an exposure-response relationship between phthalates and endocrine hormones essential for ovulation among US women. The objective of this research is to determine the relationship between levels of gonadotropins, follicle stimulating hormone (FSH) and leutinizing hormone (LH), and urinary phthalate monoester metabolites: MBP, MEP, MEHP, MBZP among National Health and Nutrition Examination Survey (NHANES) 1999-2002 women aged 35 to 60 years. Using biomarker data from a one-third sub-sample of NHANES participants, log transformed serum FSH and serum LH, respectively were regressed on phthalates controlling for age, body mass index, smoking, and creatinine taking into consideration the complex survey design (n=385). Models were stratified by reproductive status: reproductive (n=185), menopause transition (n=49) and post-menopausal (n=125). A decrease in FSH associated with increasing MBzP (beta=-0.094, p<0.05) was observed for all participants but no statistical association between log FSH and MBP, MEP, or MEHP was seen. A decrease in LH (beta=-0.125, p<0.05) was also observed with increasing MBzP for all participants though there was no relationship between levels of LH and MBP, MEP, or MEHP. The observed associations between FSH, LH and MBzP did not persist when stratified by reproductive status. Thus, the present study shows a change in endocrine hormones related to ovulation with increasing urinary MBzP among a representative sample of US women from 1999-2002 though this observed exposure-response relationship does not remain after stratification by reproductive status. ^