Inhibition of fertilization in cattle by passive immunization with anti-zona pellucida serum

Passive immunization with antiserum prepared against isolated bovine zonae pellucidae inhibited fertilization in the cow. The minimum dosage of antiserum (titer 2⁷ by immunofluorescence) required for complete inhibition was 2 ml/kg of body weight.     

Effect of anti-pig zona pellucida serum on fertilization in several mammals

The effect of goat antiserum against isolated pig zonae pellucidae on fertilization in vivo was examined in the pig, cow, sheep, rabbit, rat, and mouse. As shown by indirect immunofluorescence, anti-pig zona serum reacted strongly with the zonae of pig, cow, sheep, and rabbit, but the reaction with the zonae of mouse and rat was weak. Passive immunization with anti-pig zona serum significantly, or completely, inhibited fertilization in all species. However, inhibition of fertilization was more pronounced in the pig, cow, sheep, rabbit, and mouse than in the rat. Inhibition of fertilization in the rabbit was also observed after passive immunization with antiserum absorbed with rabbit liver and kidney. All of the zonae recovered from the pig, cow, sheep, rat, and mouse after passive immunization with anti-pig zona serum exhibited strong fluorescence, regardless of the incidence of fertilization. It was concluded that the pig and other mammalian zonae pellucidae tested have tissue-specific antigens.     

Effect of antibody to detergent solubilized zonae pellucidae on in vivo and in vitro fertilization in the mouse

An antiserum was produced in one rabbit against mouse zonae pellucidae solubilized with 70 mM Na₂SO₃, 1% SDS, and 0.04 mM CuSO₄. An IgG of zona antibody completely inhibited fertilization both in vitro and in vivo in the mouse. F(ab′)₂ fragment obtained by pepsin digestion of IgG zona antibody inhibited fertilizability of eggs in vitro but did not inhibit fertilization in vivo after passive immunization.     

Use of ultrasound to monitor prenatal growth and development in the common marmoset (Callithrix jacchus)

The increasing use of non-human primates to study fetal development and neonatal management has necessitated the availability of fetuses of known gestational history. In this study, prenatal development and growth were investigated in the common marmoset (Callithrix jacchus) using ultrasound. The objectives of this study were: (1) to determine the accuracy of ultrasound for monitoring prenatal growth and development in common marmosets, (2) to determine if litter size influences prenatal growth trajectories, and (3) to assess growth discordancy among litter mates. Fifty pregnancies were monitored longitudinally using real-time abdominal sonography. During each examination the number of fetuses was recorded, and crown-rump length (CRL) and biparietal diameter (BPD) were measured. The results indicate that ultrasound is a reliable method for observation of gross morphological changes during prenatal development in this species. Measures of CRL and BPD taken early in gestation using ultrasound were in agreement with those from gross specimens. Triplets were significantly (P < 0.05) smaller than twins for both BPD and CRL. No significant relationship was found between litter size and within litter variation in CRL or BPD. This study is the first longitudinal investigation of prenatal growth and development in C. jacchus. The observations from this study will be of use for determining approximate gestational age of fetuses, as well as providing guidelines for routine monitoring of pregnancy in this species. © 1995 Wiley-Liss, Inc.     

Characterization of the guanidinobenzoatase in the epididymal fluids of the mouse

Guanidinobenzoatase (GB), a proteolytic enzyme found in the epididymal fluids of mice, was purified to apparent homogeneity by molecular sieving and affinity chromatography. It has a molecular mass of 71 kDa and its enzymatic activity is heat labile and sensitive to EGTA. Its kinetic parameters (Km of 6.66 μM and a Vmax of 4.38 nmol/min/mg) were determined using 4-methylumbelliferyl-p-guanidinobenzoate (MUGB) as the substrate. GB activity is concentrated in the cauda epididymal region of the genital tract. Heat-solubilized whole zonae, biologically active ZP3, and several serine proteinase inhibitors, including a proteinase inhibitor endogenous to the male genital tract, effectively block the ability of GB to hydrolyze MUGB. Pretreating cumulus-free, zonae intact oocytes with purified GB reduces, in a concentration-dependent manner, the number of sperm able to bind to the zonae. The function of the soluble enzyme is not known. Its ability to bind both trypsin inhibitors and ZP3 suggests a possible role in gamete recognition. Mol. Reprod. Dev. 47:204–209, 1997. © 1997 Wiley-Liss, Inc.     

Imitation as faithful copying of a novel technique in marmoset monkeys

Imitative learning has received great attention due to its supposed role in the development of culture and the cognitive demands it poses on the individual. Evidence for imitation in non-human primate species, therefore, could shed light on the early origins of proto-cultural traits in the primate order. Imitation has been defined as the learning of an act by seeing it done or, more specifically, as the copying of a novel or otherwise improbable act. But despite a century of research and the detection of mirror neurons the empirical basis for this most advanced form of observational learning is weak. Few, if any, studies have shown that the observer has learned the response topography, i.e., the specific action by which the response is made. In an experimental set-up we confronted marmoset monkeys (Callithrix jacchus) with a conspecific model that was previously trained to open a plastic box in a peculiar way. Employing detailed motion analyses we show that the observers precisely copied the movement patterns of the novel action demonstrated by the model. A discriminant analysis classified 13 out of 14 observer movements (92.86%) as model movements and only one as non-observer movement. This evidence of imitation in non-human primates questions the dominant opinion that imitation is a human-specific ability. Furthermore, the high matching degree suggests that marmosets possess the neuronal mechanism to code the actions of others and to map them onto their own motor repertoire, rather than priming existing motor-templates.     

Stability and change in the vocal signatures of common marmoset mobbing calls

Alarm calls that carry information about the identity of the caller may help the receiver decide how to react. We recorded the tsik calls of six captive adult marmosets (Callithrix jacchus) in two different social groups in response to snake models at two time points (summer 2014 and January 2015). We measured eight acoustic variables including duration, inter-call interval, minimum and maximum frequency, and starting, maximum and ending peak frequency. Discriminant function analyses (DFA) confirmed that calls were individually distinct at both time periods (78.88 and 79.89% correctly classified at time one and time two, respectively). Stability of the vocal signatures was assessed using the DFA model for summer 2014 to classify calls elicited in January 2015. Although the classification rates were lower in 2015, calls were still classified more than would be expected by chance (64.50%). This suggests that acoustic signatures of common marmoset tsik calls remain fairly stable over time and therefore remain recognizable by their groupmates. However, during that six-month period, at least three (out of seven) acoustic parameters changed such that they were significantly higher or lower in all six marmosets; in two marmosets six out of seven parameters changed. Changes to individual ‘voices’ of animals, despite overall stability reflected in above-chance matching of calls over time in a DFA analysis, may have implications for acoustic research.     

Validation of a specific radioimmunoassay to measure plasma cortisol in the fetal sheep.

A procedure utilizing co-chromatography and complementary antiserum comparisons was employed to assess the specificity of a cortisol radioimmunoassay for use in the chronically catheterized fetal sheep preparation. Complementary antiserum comparisons is a technique by which two different cortisol antisera, prepared from conjugates attached at opposite ends of the cortisol molecule, were used to determine cortisol concentrations in the same ovine fetal plasma specimens. Results were not significantly different between the two groups, each measured by a different antiserum. This procedure may be used to assess assay specificity in any species in which steroid radioimmunoassays are being adapted.     

Response of monkeys to porcine zona pellucida as detected by a solid-phase radioimmunoassay

The immunological response of cynomolgus monkeys (Macaca fascicularis) to immunization was evaluated utilizing collagenase-isolated pig zona pellucida. Six weeks after initial immunization a high serum titer of antibody was exhibited. Serum antibody titers demonstrated a noticeable decline 5 months after booster injections were discontinued. The assay method used is rapid and is capable of detecting antibody in serum dilutions of 1:78,000, as compared to 1:125,000 with the indirect fluorescence assay.     

Use of a facemask to protect the intra-oral area in cynomolgus monkeys

A custom fiberglass facemask was designed for cynomolgus monkeys to protect an orthodontic appliance. The mask was constructed from impressions and models made of the animal's head. It prevented the fingers from entering the mouth to dislodge the intra-oral appliances. The facemask permitted normal physical activity, eating, and drinking.     

Induction of metallothionein in rat tissues following subchronic exposure to mercury shown by radioimmunoassay

Although the analysis of metallothionein (MT) by radioimmunoassay (RIA) is not a common technique, its use is preferred over other methods since it offers the advantages of sensitivity and specificity. In this paper we present data on the basal levels of MT in rat tissues and physiological fluids of female Sprague-Dawley rats. The mean basal MT concentrations of the following organs and fluids were determined by RIA to be: liver (9.8 μg/g), kidney (68 μ/g), brain (0.8 μg/g), spleen (1.0 μg/g), heart (5.4 μg/g), plasma (11 ng/ml), and urine (200–300 μg/g creatinine). Following subcutaneous exposure to inorganic mercury (0.2 μmol/kg/d, 5 d a week for up to 4 wk), the metal accumulated primarily in the kidney. There was also a simultaneous accumulation of zinc in the liver and of zinc and copper in the kidney. Induction of MT did take place in liver, kidney, brain, and spleen. No increases in the MT contents of blood and urine were noted. The excess zinc and copper in the kidney of exposed animals were found to be associated predominantly with MT. No overt signs of mercury toxicity were noted in these animals and the incidence of proteinurea was nil. The data are discussed with reference to methods of MT determination in animal tissues and in relation to mercury metabolism and toxicity.     

A radioimmunoassay for dehydroepiandrosterone sulfate in the circulation of rhesus monkeys

A radioimmunoassay for dehydroepiandrosterone sulfate (DHAS) was established and validated for use in the rhesus monkey. The validation demonstrated the co-migration of immunoreactive material with tritiated dehydroepiandrosterone after hydrolysis and indicated the absence of other interfering steroids in the measurement. The application of this assay to perinatal samples verified that there are microgram quantities of DHAS present in the circulation. The measurement of circulating concentrations of DHAS in male and female rhesus monkeys at different stages of development demonstrated the absence of increases associated with puberty in this macaque species. Concentrations of DHAS were similar in adult males and females, but were elevated in pregnant females (p less than 0.05). Adult males had increased DHAS concentrations after GnRH administration (p less than 0.05), but no change was detected in infant male monkeys. Cortisol and DHAS responses of both infant and adult males occurred at a similar dose of ACTH (0.05 mU per kg versus 0.015 mU per kg, infant and adult, respectively). These data demonstrate the validity of the DHAS measurement in the rhesus monkey and suggest that the secretion of DHAS from infant and adult adrenals is generated by a similar stimulus. Since there was no evidence of gonadal secretion of DHAS in the infant, changes in either adrenal secretion and/or metabolic clearance of DHAS probably account for the microgram concentrations found during the perinatal period.     

A comparison of chemiluminescence immunoassay and radioimmunoassay for the detection and quantification of methyltestosterone residues in meat samples

A chemiluminescence immunoassay (CLIA) for methyltestosterone is compared with a radioimmunoassay (RIA) employing the same antiserum, raised against methyltestosterone-3-CMO-BSA and using N-(4-aminobutyl)-N-ethylisoluminol conjugate of MT and [1,2-H³]methyltestosterone as tracers. Muscle tissue from slaughtered animals was selected as the matrix. After enzymatic digestion and diethylether extraction only a limited sample clean-up on Lipidex-5000 and on Bond Elut C18 was required. Both methods had similar limits of detection, sensitivities, limits of quantification and speed of analysis (working-load and availability of results).     

Fecal cortisol radioimmunoassay to monitor adrenal gland activity in the bottlenose dolphin (Tursiops truncatus) under human care

Fecal glucocorticoid measurement is an important noninvasive tool to monitor animal health. A radioimmunoassay (RIA) method was developed to measure fecal cortisol in bottlenose dolphins under human care. The method was used to measure baseline hormone levels and evaluate the adrenal response to environmental challenges in a small number of individual dolphins. The method was validated by precision and accuracy tests and by comparison with liquid chromatography‐mass spectrometry (LC‐MS). The parallelism test suggested few matrix interferences. The assay showed a good degree of precision within assay (CV = 5.4%) and between assays (CV = 4.1%). The RIA significantly correlated with the LC‐MS method (r = 0.838, P < 0.01). The recovery test and the comparison between RIA and LC‐MS suggested that the RIA slightly underestimates fecal cortisol concentrations, although the degree of accuracy was good. This study established that bottlenose dolphins excrete appreciable amounts of fecal cortisol (healthy subjects: 0.2–9.5 ng/g). Therefore, chronic HPA axis activation may be monitored in fecal samples by immunoassays after validating a suitable extraction protocol. The RIA could discriminate conditions of stimulation (pregnancy, parturition, isolation, transportation) and inhibition (diazepam administration) of the HPA axis and may, therefore, be useful for monitoring dolphin well‐being.     

Development of a computational technique to measure cartilage contact area

Computational measurement of joint contact distributions offers the benefit of non-invasive measurements of joint contact without the use of interpositional sensors or casting materials. This paper describes a technique for indirectly measuring joint contact based on overlapping of articular cartilage computer models derived from CT images and positioned using in vitro motion capture data. The accuracy of this technique when using the physiological nonuniform cartilage thickness distribution, or simplified uniform cartilage thickness distributions, is quantified through comparison with direct measurements of contact area made using a casting technique. The efficacy of using indirect contact measurement techniques for measuring the changes in contact area resulting from hemiarthroplasty at the elbow is also quantified. Using the physiological nonuniform cartilage thickness distribution reliably measured contact area (ICC=0.727), but not better than the assumed bone specific uniform cartilage thicknesses (ICC=0.673). When a contact pattern agreement score (s_agree) was used to assess the accuracy of cartilage contact measurements made using physiological nonuniform or simplified uniform cartilage thickness distributions in terms of size, shape and location, their accuracies were not significantly different (p>0.05). The results of this study demonstrate that cartilage contact can be measured indirectly based on the overlapping of cartilage contact models. However, the results also suggest that in some situations, inter-bone distance measurement and an assumed cartilage thickness may suffice for predicting joint contact patterns.     

Enzymic synthesis of corynanthe-type alkaloids in cell cultures of Catharanthus roseus: Quantitation by radioimmunoassay

The soluble enzyme system from Catharanthus roseus cell suspension cultures which synthesizes ajmalicine, 19-epiajmalicine and tetrahydroalstonine from tryptamine and secologanin has been further characterized. The enzymic reaction was followed quantitatively by using a radioimmunoassay (RIA) with antibodies directed against ajmalicine. This RIA proved exceedingly useful in determining the enzymology of the reaction and displayed a sensitivity shown previously only by radio-tracer methods. By this method, the enzyme system was found to function optimally at pH 6.5. Sensitivity of the enzymic synthesis of ajmalicine and its stereoisomers to δ-d-gluconolactone, a β-glucosidase inhibitor, indicated the involvement of a β-glucosidase in the formation of these alkaloids. The enzyme system catalysed the formation of unnatural ajmalicine analogues from ring-substituted tryptamines.     

A radioimmunoassay specific for [Gln]LH-RH: Application in the confirmation of the structure of chicken hypothalamic luteinizing hormone-releasing hormone

In the first report on the chemical structure of a nonmammalian LH-RH, chicken hypothalamic LH-RH was demonstrated to be [Gln⁸]LH-RH [2–4]. However, these studies and subsequent reports [7,8] did not totally exclude the possibility of a reverse sequence of the two amino acids Leu-Gln. In view of the recently described structure of salmon brain LH-RH as [Trp⁷,Leu⁸]LH-RH [9], we undertook to confirm our earlier conclusion that chicken LH-RH is [Gln⁸]LH-RH and not [Gln⁷,Leu⁸]LH-RH. The immunologic, chromatographic and biological properties of natural chicken hypothalamic LH-RH were compared with those of the two synthetic peptides, [Gln⁸]LH-RH and [Gln⁷,Leu⁸]LH-RH. A radioimmunoassay highly specific for [Gln⁸]LH-RH was developed. Natural chicken LH-RH cross-reacted fully with the antiserum which requires the COOH-terminal Gln⁸ to Gly¹⁰-NH₂ for binding, while [Gln⁷,Leu⁸]LH-RH showed less than 0.1% cross-reaction. On a high resolution reverse phase high performance liquid chromatography system, natural chicken LH-RH co-eluted with [Gln⁸]LH-RH and was well separated from [Gln⁷,Leu⁸]LH-RH. In a chicken anterior pituitary cell bioassay, natural chicken LH-RH and [Gln⁸]LH-RH were equipotent in stimulating luteinizing hormone release, while the relative potency of [Gln⁷,Leu⁸]LH-RH was 4.4%. These data, in particular the use of a specific [Gln⁸]LH-RH antiserum, provide conclusive evidence that chicken LH-RH is [Gln⁸]LH-RH.