Cyclic mRNA expression of thyrotropin subunits and deiodinases in red drum, Sciaenops ocellatus.

The role of thyrotropin (thyroid-stimulating hormone, TSH) in driving peripheral thyroid function in non-mammalian species is still poorly understood. Thyroxine (T4), the principal hormone released from the thyroid gland in response to TSH stimulation, circulates with a robust daily rhythm in the teleost fish the red drum. Previous research suggests that the red drum T4 cycle is circadian in nature, driven by TSH secretion in the early photophase and inhibited by T4 feedback in the early scotophase. To determine whether TSH is produced in a pattern consistent with feedback inhibition by this T4 cycle, we used quantitative real time PCR (qPCR) to quantify the daily cycle of expression of the pituitary TSH subunits GSUα, and TSHß. We found that TSH expression cycled inversely to, and 6-12 h out of phase with, the T4 cycle, consistent with the hypothesis that TSH secretion drives the T4 cycle. To examine the potential role of deiodinases in negative feedback regulation of this TSH cycle, we also utilized qPCR to assess the pituitary expression patterns of the TH activating enzyme outer-ring deiodinase (Dio2) and the TH deactivating enzyme inner-ring deiodinase (Dio3). Dio2 was not expressed with an obvious daily cycle, whereas Dio3 expression mirrored the expression of TSH. These results are consistent with circulating T4 providing the negative feedback signal controlling both TSH production and Dio3 expression in the pituitary, and suggest that TH inactivation by inner ring deiodination is an important component of TSH negative feedback control.

Thyrotropic activity of recombinant human glycoprotein hormone analogs and pituitary mammalian gonadotropins in goldfish (Carassius auratus): insights into the evolution of thyrotropin receptor specificity.

Thyrotropin (TSH) is a pituitary glycoprotein hormone heterodimer that binds to its G-protein coupled receptor (TSH-R) at the thyroid to promote the synthesis and secretion of thyroid hormone. Very little is known about TSH-TSH-R interactions in teleost fish. Mammalian gonadotropins have been reported to have an intrinsic ability to activate teleost fish TSH-Rs, suggesting the TSH-R in teleost fish is more promiscuous than in other vertebrates. In this study we utilized the goldfish T(4)-release response and recombinant human TSH analogs as in vivo tools to evaluate the structural constraints on hormone-receptor interactions. We found that four positively charged lysines substituted for neutral or negatively charged amino acids within positions 11-20 of the glycoprotein hormone subunit α (GSUα) significantly increased biological activity of hTSH in fish, as it does in mammals. We further found that bovine follicle stimulating hormone but not luteinizing hormone, whose GSUα subunits also contain four lysine or arginine amino acid residues in the N-terminal portion of GSUα, was thyrotropic in goldfish, suggesting gonadotropin ß subunit contributes to the heterothyrotropic activity. Though recombinant human FSH did not produce a dose-dependent increase in T(4), thyrotropic activity could be acquired with the addition of positively charged amino acids at the N-terminal portion of its GSUα, confirming the importance of the charge on those amino acids for activation of the goldfish TSH-R. These studies demonstrate that mammalian glycoprotein hormone analogs can be utilized to evaluate the conservation of receptor binding and activation mechanisms between fish and mammals.

Early pregnancy detection and the hormonal characterization of embryonic-fetal mortality in fallow deer (Dama dama).

The objectives of this investigation were to 1) determine serum concentrations of progesterone (P4), estrone sulfate (E1S) and pregnancy-specific protein B (PSPB) from estrus synchronization through mid-gestation in the fallow doe (Dama dama) and 2) characterize the hormonal profiles of does whose embryos or fetuses died in utero. Ten fallow does were synchronized for 14 d with an intravaginal P4-releasing device (CIDR) and were naturally mated after CIDR removal. Blood samples were collected at CIDR insertion, CIDR removal and at intervals through Day 203 post-CIDR removal for analysis of P4, E1S and PSPB by radioimmunoassay (RIA). Ultrasonography was performed on Days 49 and 69 post-CIDR removal. Serum P4 at the time of CIDR insertion was 4.8 +/- 0.6 ng/ml, and at CIDR withdrawal it was 6.2 +/- 0.3 ng/ml. Concentrations of E1S and PSPB were nondetectable at CIDR insertion. Serum E1S was highest at Day 93, and PSPB was first detectable in pregnant does at Days 27 to 30 post-CIDR withdrawal. Ultrasonography on Day 49 revealed that 6 does were pregnant, 2 were not pregnant and 2 others were diagnosed originally as early pregnant. At Day 69, ultrasonography revealed that 6 does (60%) were pregnant and 4 (40%) were not. A comparison of the ultrasonographic and hormonal data indicated that the 2 does diagnosed as early pregnant on Day 49 had conceived but had lost the pregnancy. A third doe which was pregnant on Day 69 lost the fetus later in gestation. Hormonal profiles of does whose embryo or fetus had died were characterized by erratic P4 and E1S profiles, with PSPB becoming undetectable in the 3 does by Days 49, 65 and 80 post-CIDR removal. These data 1) demonstrate the timing for the collection of serum samples for determining early pregnancy in fallow does using 3 hormonal methods and 2) characterize the hormonal profiles of 3 fallow does with embryonic-fetal loss.

Reproductive effects of fenoxycarb on sheep.

Fenoxycarb (ethyl [2-(4-phenoxyphenoxy)-ethyl] carbamate) is an insect growth regulator used for long-term fire ant control. Because of its effects on insect reproduction and its potential use on pasturage consumed by food animals, a reproductive study was conducted using Rambouillet sheep. The sheep were dosed daily with a placebo or with fenoxycarb at 0.69 or 1.38 mg/kg/day, representing ten (10x) and 20 times (20x) the maximum amounts of fenoxycarb in forage or hay treated at recommended levels for fire ant control. Parameters that were measured included rates of weight gain of adults, serum clinical chemistry profiles of adults, spermatozoal morphology and motility, estrus cycling, pregnancy rates, maintenance of pregnancies to term, numbers of live births, and rates of weight gain of lambs to 28 days. There were no statistically significant (P < or = 0.05) differences between the exposed and control groups of sheep in any of these facets of the study. No clinical signs associated with exposure to fenoxycarb were observed in any animal at any time, and no exposure-related pattern of pathologic lesions or reproductive organ histology was observed. Means of hepatic fenoxycarb residues in the rams followed a statistically significant (P < or = 0.05) dose-related pattern. No fenoxycarb was detected (detection limit of 5 ppb) in any neonatal liver, despite the presence of hepatic fenoxycarb residues in the treated ewes, indicating that transplacental transport of fenoxycarb was minimal. No fenoxycarb was detected in any lamb liver at 28 days, although both the colostrum and the milk of exposed ewes were found to contain fenoxycarb at levels proportional to the treatments. Based on the lack of significant findings in this study, it is unlikely that use of fenoxycarb, according to label instructions (currently applicable to homeowner and registered agricultural usage) for fire ant control in pasturage or hay fields, will affect ruminant reproduction.

Artificial insemination, hybridization and pregnancy detection in sika deer (Cervus nippon ).

Artificial insemination (AI) was performed on sika hinds (Cervus nippon ) receiving various dosages of pregnant mare serum gonadotropin (PMSG; Year 1: 0, 50 and 100 IU; Year 2: 100 and 150 IU) and using semen collected from elk and 1 2 elk x 1 2 sika stags. The time from synchronization device removal (CIDR vs norgestomet) to estrus was determined through observations of mounting activity. Methods for pregnancy detection, serum progesterone (P4), estrone sulfate (E1S), pregnancy-specific protein B (PSPB) and ultrasonography, following AI (Year 1: AI, Days 28 and 48 after AI; Year 2: AI, Days 42, 53 and 100 after AI) and a 90-d natural breeding season were investigated. From available production data, body weights were compared among sika and 1 4 elk x 3 4 sika hybrids relative to age. Pregnancy rates tended (P < 0.10) to differ relative to PMSG treatment and sire; administration of 0 IU PMSG resulted in fewer hinds becoming pregnant to AI than 50 or 100 IU of PMSG. Hinds receiving 100 IU of PMSG had higher (P < 0.05) pregnancy rates than hinds receiving 150 IU PMSG. Time to standing estrus did not differ (P > 0.10) between the CIDR and norgestomet groups. Pregnancy rates 50 d after a 90-d breeding season were similar (P > 0.10) between ultrasound (70.9%) and PSPB (61.6%). Serum P4 after 90 d in breeding groups and 50 d after stag removal were higher (P < 0.05) for pregnant than open hinds. Pregnancy rates (Year 1) 48 d after AI were similar (P > 0.10) between ultrasound (49.0%) and PSPB (37.3%). Serum P4 28 and 48 d after AI were higher (P < 0.05) for pregnant than open hinds. Serum E1S was higher (P < 0.01) for pregnant than open hinds 48 d after AI. Pregnancy rates (Year 2) 100 d after AI did not differ (P > 0.10) between ultrasound and PSPB (66.7%). Serum P4 was higher (P < 0.03) in pregnant than open hinds at 42, 53 and 100 d after AI. At 100 d after AI, pregnant hinds had higher (P < 0.002) serum E1S than open hinds. At 6 to 8 and 11 to 13 mo of age, 1 4 elk x 3 4 sika males tended (P < 0.08) to be heavier than sika males, while 1 4 elk x 3 4 sika females were heavier (P < 0.05) than sika females at all ages. In summary, this study documents the use of AI and methods for pregnancy detection in sika hinds as well as preliminary information regarding the production of elk-x-sika hybrids.

Methods for pregnancy determination and the effects of body condition on pregnancy status in Rocky mountain elk (Cervus elephus nelsoni ).

The objective of this study was to determine the effectiveness of transrectal ultrasonography and serum progesterone (P(4)), estrone sulfate (E(1)S) and pregnancy-specific protein B (PSPB), without prior knowledge of reproductive status, in detecting pregnancy in elk cows. In addition, body weight and body condition score (BCS) were determined to assess whether body condition affects pregnancy status in elk cows. Twenty-five elk cows were sampled during the early rut (Period 1) and after the rut (Period 2), an interval of 120 d. Age, weight, BCS and blood samples, for P(4), E(1)S and PSPB determinations, were taken at Periods 1 and 2. Ultrasonography was performed at Period 2. The younger elk cows weighed less (P<0.05) than older cows. However, pregnancy status was not affected (P> 0.10) by age or weight of the cow. Elk cows that calved had higher (P<0.02) BCS at Periods 1 and 2 than cows that remained open. Serum P(4) and E(1)S were higher (P<0.0001) in pregnant cows at Period 2 than in open cows. Progesterone was 85.8% accurate in detecting pregnant versus open cows at Period 1, while E(1)S and PSPB were not effective. Elk cows at Period 1 were <20 d pregnant with the exception of 1 cow at 46 d. Ultrasonography was 92% accurate, P(4) was 95% accurate, and E(1)S and PSPB were both 100% accurate in determining pregnant versus open cows at Period 2. Pregnant cows at Period 2 were all > 100 d pregnant. Ultrasonography, serum E(1)S and PSPB all may provide a reliable means for pregnancy diagnosis in elk cows at > 100 d of gestation, while serum P(4) may be effective when multiple samples are compared during or after the rut, or when used in combination with the other diagnostic methods described. Further research is needed to determine the optimum time period after breeding in elk cows for accurate pregnancy detection through hormonal analysis.

Estimation of genotype distributions and posterior genotype probabilities for beta-mannosidosis in Salers cattle.

beta-Mannosidosis is a lethal lysosomal storage disease inherited as an autosomal recessive in man, cattle and goats. Laboratory assay data of plasma beta-mannosidase activity represent a mixture of homozygous normal and carrier genotype distributions in a proportion determined by genotype frequency. A maximum likelihood approach employing data transformations for each genotype distribution and assuming a diallelic model of inheritance is described. Estimates of the transformation and genotype distribution parameters, gene frequency, genotype fitness and carrier probability were obtained simultaneously from a sample of 2,812 observations on U.S. purebred Salers cattle with enzyme activity, age, gender, month of pregnancy, month of testing, and parents identified. Transformations to normality were not required, estimated gene and carrier genotype frequencies of 0.074 and 0.148 were high, and the estimated relative fitness of heterozygotes was 1.36. The apparent overdominance in fitness may be due to a nonrandom sampling of progeny genotypes within families. The mean of plasma enzyme activity was higher for males than females, higher in winter months, lower in summer months and decreased with increased age. Estimates of carrier probabilities indicate that the test is most effective when animals are sampled as calves, although effectiveness of the plasma assay was less for males than females. Test effectiveness was enhanced through averaging repeated assays of enzyme activity on each animal. Our approach contributes to medical diagnostics in several ways. Rather than assume underlying normality for the distributions comprising the mixture, we estimate transformations to normality for each genotype distribution simultaneously with all other model parameters. This process also excludes potential biases due to data preadjustment for systematic effects. We also provide a method for partitioning phenotypic variation within each genotypic distribution which allows an assessment of the value of repeat measurements of the predictive variable for genotype assignment.