Hormonal priming, induction of ovulation and in-vitro fertilization of the endangered Wyoming toad (Bufo baxteri)

Abstract The endangered Wyoming toad (Bufo baxteri) is the subject of an extensive captive breeding and reintroduction program. Wyoming toads in captivity rarely ovulate spontaneously and hormonal induction is used to ovulate females or to stimulate spermiation in males. With hormonal induction, ovulation is unreliable and egg numbers are low. The sequential administration of anovulatory doses of hormones (priming) has increased egg numbers and quality in both anurans and fish. Consequently, we tested the efficacy of a combination of human Chorionic Gonadotrophin (hCG) and Luteinizing Hormone Releasing Hormone analogue (LHRHa) administered as one dose, or two or three sequential doses to Bufo baxteri on egg numbers, fertilization and early embryo development. Spawning toads deposited eggs into Simplified Amphibian Ringers (SAR) solution to enable controlled in-vitro fertilization (IVF) with sperm from hormonally induced male toads. Unprimed females receiving a single mixed normally ovulatory dose of 500 IU hCG plus 4 micrograms of LHRHa produced no eggs. Whereas females primed with this dose and an anovulatory dose (100 IU hCG and 0.8 micrograms of LHRHa) of the same hormones, or primed only with an anovulatory dose, spawned after then receiving an ovulatory dose. Higher total egg numbers were produced with two primings than with one priming. Moreover, two primings produced significantly more eggs from each individual female than one priming. The cleavage rate of eggs was not found to differ between one or two primings. Nevertheless, embryo development with eggs from two primings gave a significantly greater percentage neurulation and swim-up than those from one priming. Of the male toads receiving a single dose of 300 IU hCG, 80% produced spermic urine with the greatest sperm concentration 7 hours post-administration (PA). However, peak sperm motility (95%) was achieved at 5 hours PA and remained relatively constant until declining 20 hours PA. In conclusion, Bufo baxteri egg numbers and quality benefited from sequential priming with LHRHa and hCG whereas spermic urine for IVF was produced from males with a single dose of hCG. The power of assisted reproduction technology in the conservation of endangered amphibians is shown by the release of nearly 2000 tadpoles produced by IVF during this study.</p

Frequency of exogenous hormone therapy impacts spermiation in male Fowler\u27s toad (Bufo fowleri)

Amphibians are experiencing a global extinction crisis and captive assurance colonies, along with reintroduction programs, are necessary to prevent further losses. Assisted reproductive technologies (ART), such as hormone-stimulated gamete collection and in vitro fertlisation (IVF), are conservation methods that can be used to increase reproductive output for breeding and reintroduction programs when animals fail to breed naturally. In order to maximise the production of offspring using ART, it is important to establish the physiological limitations on the frequency that hormone therapy can be used to collect gametes for IVF or assisted breeding. The present study examined the effects of the frequency of hormone-induced spermiation on sperm quantity and quality in Fowler\u27s toad (Bufo fowleri) by comparing four levels of hormone injection frequencies: twice a week, once a week, every other week, and every 3 weeks. Sperm release was induced with an intraperitoneal injection of 300IU human chorionic gonadotropin (hCG). Spermatozoa were collected at three time points after injection (5, 7 and 9h) and sperm concentration, motility and quality of forward progressive movement were measured. A significant decrease in sperm concentration (P\u3c0.01) was observed with the most frequent treatment (twice a week hormone injections). However, there was no negative effect of the treatments on sperm motility (P≤0.06) or forward movement (P≤0.06). We also observed a significant decrease in the concentration (P\u3c0.01), motility (P≤0.02) and quality of forward progressive movement (P≤0.01) of spermatozoa at the 9h collection compared with earlier collection times. These results have clear implications for amphibian captive breeding programs, where more frequent hormone-induced spermiation could have a negative effect on male performance. We recommend that hormone injections be spaced a minimum of 2 weeks apart to optimise the health of the animals, assisted breeding, IVF or collection of gametes for genome resource banking

Bovine Respiratory Syncytial Virus (BRSV) Infection Detected in Exhaled Breath Condensate of Dairy Calves by Near-Infrared Aquaphotomics

Bovine respiratory syncytial virus (BRSV) is a major contributor to respiratory disease in cattle worldwide. Traditionally, BRSV infection is detected based on non-specific clinical signs, followed by reverse transcriptase-polymerase chain reaction (RT-PCR), the results of which can take days to obtain. Near-infrared aquaphotomics evaluation based on biochemical information from biofluids has the potential to support the rapid identification of BRSV infection in the field. This study evaluated NIR spectra (n = 240) of exhaled breath condensate (EBC) from dairy calves (n = 5) undergoing a controlled infection with BRSV. Changes in the organization of the aqueous phase of EBC during the baseline (pre-infection) and infected (post-infection and clinically abnormal) stages were found in the WAMACS (water matrix coordinates) C1, C5, C9, and C11, likely associated with volatile and non-volatile compounds in EBC. The discrimination of these chemical profiles by PCA-LDA models differentiated samples collected during the baseline and infected stages with an accuracy, sensitivity, and specificity &gt;93% in both the calibration and validation. Thus, biochemical changes occurring during BRSV infection can be detected and evaluated with NIR-aquaphotomics in EBC. These findings form the foundation for developing an innovative, non-invasive, and in-field diagnostic tool to identify BRSV infection in cattle

Profiling Mannheimia haemolytica infection in dairy calves using near infrared spectroscopy (NIRS) and multivariate analysis (MVA)

Abstract Bovine respiratory disease (BRD) linked with Mannheimia haemolytica is the principal cause of pneumonia in cattle. Diagnosis of BRD traditionally relies on visual assessment, which can be untimely, insensitive, and nonspecific leading to inadequate treatment and further spread of disease. Near Infrared Spectroscopy (NIRS) is a rapid acquisition vibrational spectroscopy that can profile changes in biofluids, and when used in combination with multivariate analysis, has potential for disease diagnosis. This study characterizes the NIR spectral profile of blood plasma from dairy calves infected with M. haemolytica and validates the spectral biochemistry using standardized clinical and hematological reference parameters. Blood samples were collected for four days prior to (baseline), and 23 days after, a controlled intrabronchial challenge. NIR spectral profiles of blood plasma discriminated and predicted Baseline and Infected states of animal disease progression with accuracy, sensitivity, and specificity ≥ 90% using PCA–LDA models. These results show that physiological and biochemical changes occurring in the bloodstream of dairy calves during M. haemolytica infection are reflected in the NIR spectral profiles, demonstrating the potential of NIRS as a diagnostic and monitoring tool of BRD over time

Phenological changes in bamboo carbohydrates explain the preference for culm over leaves by giant pandas (Ailuropoda melanoleuca) during spring.

Seasonal changes in the foodscape force herbivores to select different plant species or plant parts to meet nutritional requirements. We examined whether the search for calorie-rich carbohydrates explained giant panda's selection for bamboo culm over leaves during spring. Leaves and culms were collected from four Phyllostachys bamboos (P. aurea, P. aureosulcata, P. glauca, and P. nuda) once per month over 18-27 months. Monthly changes in annual plant part nutrients were examined, and compared to seasonal foraging behaviors of captive giant pandas. Although total fiber was greater (p<0.0001) in culm (85.6 ± 0.5%) than leaves (55.3 ± 0.4%) throughout the year, culm fiber was at its lowest in spring (79-85%) when culm selection by giant pandas exceeded 70% of their overall diet. Culm starch also was greatest (p = 0.044) during spring (5.5 ± 1.1%) and 2.5-fold the percentage of starch in leaves (2.2 ± 0.6%). The free sugars in spring culm consisted of a high proportion of glucose (35%) and fructose (47%), whereas sucrose made up 42% of the total free sugar content of spring leaves. Bound sugars in culm consisted of 60% glucose and 38% xylose likely representative of hemicellulose. The concentrations of bound sugars (hemicelluloses) in spring culms (543.7 ± 13.0 mg/g) was greater (p<0.001) than in leaves (373.0 ± 14.8 mg/g). These data help explain a long-standing question in giant panda foraging ecology: why consume the plant part with the lowest protein and fat during the energetically intensive spring breeding season? Giant pandas likely prefer spring culm that contains abundant mono- and polysaccharides made more bioavailable as a result of reduced fiber content. These data suggest that phenological changes in bamboo plant part nutrition drive foraging decisions by giant pandas

Near-Infrared Spectroscopy (NIRS) as a Method for Biological Sex Discrimination in the Endangered Houston Toad (<i>Anaxyrus houstonensis</i>)

Biological sex is one of the more critically important physiological parameters needed for managing threatened animal species because it is crucial for informing several of the management decisions surrounding conservation breeding programs. Near-infrared spectroscopy (NIRS) is a non-invasive technology that has been recently applied in the field of wildlife science to evaluate various aspects of animal physiology and may have potential as an in vivo technique for determining biological sex in live amphibian species. This study investigated whether NIRS could be used as a rapid and non-invasive method for discriminating biological sex in the endangered Houston toad (Anaxyrus houstonensis). NIR spectra (N = 396) were collected from live A. houstonensis individuals (N = 132), and distinct spectral patterns between males and females were identified using chemometrics. Linear discriminant analysis (PCA-LDA) classified the spectra from each biological sex with accuracy ≥ 98% in the calibration and internal validation datasets and 94% in the external validation process. Through the use of NIRS, we have determined that unique spectral signatures can be holistically captured in the skin of male and female anurans, bringing to light the possibility of further application of this technique for juveniles and sexually monomorphic species, whose sex designation is important for breeding-related decisions