NMR-based metabolomics of plasma from dairy calves infected with two primary causal agents of bovine respiratory disease (BRD).

Each year, bovine respiratory disease (BRD) results in significant economic loss in the cattle sector, and novel metabolic profiling for early diagnosis represents a promising tool for developing effective measures for disease management. Here, 1H-nuclear magnetic resonance (1H-NMR) spectra were used to characterize metabolites from blood plasma collected from male dairy calves (n = 10) intentionally infected with two of the main BRD causal agents, bovine respiratory syncytial virus (BRSV) and Mannheimia haemolytica (MH), to generate a well-defined metabolomic profile under controlled conditions. In response to infection, 46 metabolites (BRSV = 32, MH = 33) changed in concentration compared to the uninfected state. Fuel substrates and products exhibited a particularly strong effect, reflecting imbalances that occur during the immune response. Furthermore, 1H-NMR spectra from samples from the uninfected and infected stages were discriminated with an accuracy, sensitivity, and specificity ≥ 95% using chemometrics to model the changes associated with disease, suggesting that metabolic profiles can be used for further development, understanding, and validation of novel diagnostic tools.

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 >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).

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.

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

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.

Reproductive Performance of Mares Fed Dietary Zearalenone.

It is known that zearalenone (ZON) interacts directly with estrogen receptors, and its in vivo effects on reproduction have been well-documented in several species. In contrast, reports of ZON's impact on horse reproduction are conflicting and inconclusive, some studies confounded by the presence of mycotoxins such as deoxynivalenol in the feed. This study assesses the effect of chronic consumption of zearalenone on reproduction in cycling mares fed >95% pure ZON (0, 2, or 8 mg/da; n = 7 mares/treatment) for three estrous cycles, followed by artificial insemination, through 16 days of pregnancy. Animals were on ZON treatment for between 70 and 121 days (average 84) depending on individual cycle patterns. ZON-induced changes in serum concentration of estradiol (E2) and progesterone (P4), and total estrogenicity were measured using RIAs and the E-screen assay, respectively. Effects on reproductive physiology and pregnancy were monitored by ultrasound and clinical parameters. No significant changes were found in reproductive hormone levels of E2, or P4 for mares on ZON treatments compared to controls, although there was a significant (P < 0.01) increase in P4 levels across Cycle number in High ZON (8 mg/da) treated mares. There was also an increasing trend in the interovulatory interval in the High ZON treatment group. The overall estrogenicity was similar across treatments and over time, not differing from controls or between ZON treatment groups. Adverse uterine and ovarian effects were also not observed, but pregnancy rates were mixed with only 4 of 7 mares on Low ZON becoming pregnant, and only 3 maintaining pregnancy and fetal heartbeat by Day 30, compared to 5 of 6 control mares and all 7 mares on High ZON. Because reproductive efficiency and hormone concentrations are highly variable across individuals, this study did not demonstrate that ZON at 2 or 8 mg/da was detrimental to mares' reproduction. Yet, inferring that ZON treatments were completely without effect is also not appropriate, as the absence of measurable significant differences could be attributed to the limited sample size. Most importantly, there were no extreme signs of toxicology, in contrast to previous reports when ZON was fed at these "doses."

Ultrasound imaging improves hormone therapy strategies for induction of ovulation and in vitro fertilization in the endangered dusky gopher frog (Lithobates sevosa).

Establishing captive breeding populations of amphibians is an important conservation strategy to safeguard against ongoing declines of wild populations and provide broodstock for reintroduction programs. The endangered dusky gopher frog (DGF) has never naturally reproduced in captivity and requires breeding intervention to sustain the population. Methods for inducing ovulation in female DGFs using hormone therapies have not been evaluated. To address this need, we tested four exogenous hormone treatments to induce ovulation in DGFs (n = 11/treatment), including: treatment (A) gonadotropin-releasing hormone agonist (GnRHa); (B) GnRHa with dopamine antagonist metoclopramide hydrochloride; (C) GnRHa and human chorionic gonadotropin (hCG) and (D) GnRHa with hCG following two low hCG priming doses. Treatments B, C and D resulted in a significantly greater (P < 0.0125) number of ovulating females compared to the control (no hormone); Treatment A was not different from control. For ovulating females, the number of eggs, relative fecundity and cleavage rates of eggs were compared between the four hormone treatments and initial ultrasound grade. Between treatments, there was no difference in number of eggs or relative fecundity; however, Treatments A and D resulted in higher (P < 0.05) cleavage rates than Treatment C, but were not different from Treatment B. Ultrasound imaging was used to assess the ovarian state of DGF females prior to and following hormone therapy. A grading scale (Grades 1-5) was developed to characterize ovarian states. Ultrasound grade was found to be a significant (P = 0.002) predictor for ovulation following hormone treatment, with only high-grade females (Grades 3-4) ovulating in response to hormones. Ultrasound grade did not influence egg numbers or cleavage rate (P > 0.05). Results demonstrate multiple hormone therapies are available for stimulating ovulation in female DGFs and ultrasonography is a valuable tool to inform hormone therapy. Ultimately, these reproductive technologies are critical to enhance breeding and reintroduction efforts for the DGF.

Changes in urinary androgen concentration indicate that male giant pandas (Ailuropoda melanoleuca) respond to impending female oestrus during and outside the typical spring breeding season.

Giant pandas have been described as mono-oestrus spring breeders, yet males exposed to aseasonal oestrous females in the autumn or winter exhibit breeding behaviours and interest in mating. In the present study, urinary androgens and sperm parameters were quantified for males exposed to females expressing oestrus during spring, autumn or winter to examine plasticity of reproductive seasonality in giant pandas. Monthly average androgen concentrations for two males exposed to females in either seasonal or aseasonal oestrus were greater (P<0.001) than baseline concentrations. Evaluation of daily androgen concentrations revealed a peak that was three- to fivefold greater than baseline, occurring an average of 5 days before ovulation for both seasonal and aseasonal cycles. There were no significant differences in testes volume, sperm motility, forward progression or sperm concentration in males between female seasonal and aseasonal cycle years. Male gonadal activity was more variable without a clear pattern in years when the female was anovulatory than when she was ovulatory (seasonal or aseasonal). These data show the flexible reproductive capacity of male giant pandas as demonstrated by a rapid physiological readiness to mate in response to female oestrous cues within or outside the normal breeding season and may suggest a facultative seasonal reproduction with a 'female-induced rut'.

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.

Frequency of exogenous hormone therapy impacts spermiation in male Fowler's 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's 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<0.01) 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<0.01), 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.

Fecal estrogen, progestagen and glucocorticoid metabolites during the estrous cycle and pregnancy in the giant anteater (Myrmecophaga tridactyla): evidence for delayed implantation.

BACKGROUND: Declining numbers of wild giant anteaters highlight the importance of sustainable captive populations. Unfortunately, captive reproductive management is limited by the lack of external physical indicators of female reproductive status and the aggressive behavior of males. We examined the endocrinology of the estrous cycle and pregnancy, and whether delayed implantation is a gestational strategy for giant anteaters as described for other xenarthrans. METHODS: Feces were collected from seven captive females 3-5 times weekly and mating was recorded. Concentrations of estrogen (estrone-glucuronide, E1, and estradiol-17ß, E2), progestagen (20-oxo-progestagens, P4), and glucocorticoid (GC) metabolites were examined in fecal extracts by enzyme immunoassay. RESULTS: Estrous cycles for nulliparous females (6 cycles, n = 2) compared to the multiparous female (6 cycles, n = 1) were shorter (47.3 +/- 4.3 days versus 62.5 +/- 2.6 days) with relatively lower luteal phase concentrations of P4 (49.4 +/- 2.9 ng/g versus 136.8 +/- 1.8 ng/g). The four remaining females had unclear ovarian activity: two females exhibited apparent luteal activity but unclear fluctuations in estrogens, while the other two females had parallel fecal P4 and estrogens concentrations. Pregnancy ranged 171-183 days with females returning to estrus post-partum as early as 60 days (n = 3, 1.8-4 years of age at mating). Delayed implantation was indicated by a biphasic elevation in fecal P4 metabolites: the initial 4-fold increase occurred for 81-105 days and was followed by a 26-fold secondary rise in P4 metabolites lasting 66-94 days prior to parturition. Fecal GC was correlated with fecal estrogens and greatest during estrus, late pregnancy, and six days prior to parturition (estrous cycle GC, 14.4-62.8 ng/g; pregnancy GC, 13.6-232.7 ng/g). CONCLUSIONS: Estrous cycles of giant anteaters occurred year-round, but were shorter and more intermittent in younger nulliparous animals compared to a multiparous female. A pronounced elevation in fecal P4, estrogen, and GC occurred during late gestation after an initial post-mating delay providing evidence for delayed implantation. Adrenocorticoid activity indicated impending parturition. Differences in estrous cycle characteristics with age and the protracted but variable gestation length must be considered to improve reproductive success and neonatal survival in giant anteaters.

A comparison of human chorionic gonadotropin and luteinizing hormone releasing hormone on the induction of spermiation and amplexus in the American toad (Anaxyrus americanus).

BACKGROUND: Captive breeding programs for endangered amphibian species often utilize exogenous hormones for species that are difficult to breed. The purpose of our study was to compare the efficacy of two different hormones at various concentrations on sperm production, quantity and quality over time in order to optimize assisted breeding. METHODS: Male American toads (Anaxyrus americanus) were divided into three separate treatment groups, with animals in each group rotated through different concentrations of luteinizing hormone releasing hormone analog (LHRH; 0.1, 1.0, 4.0 and 32 micrograms/toad), human chorionic gonadotropin (hCG; 50, 100, 200, and 300 IU), or the control over 24 hours. We evaluated the number of males that respond by producing spermic urine, the sperm concentration, percent motility, and quality of forward progression. We also evaluated the effects of hCG and LHRH on reproductive behavior as assessed by amplexus. Data were analyzed using the Generalized Estimating Equations incorporating repeated measures over time and including the main effects of treatment and time, and the treatment by time interaction. RESULTS: The hormone hCG was significantly more effective at stimulating spermiation in male Anaxyrus americanus than LHRH and showed a dose-dependent response in the number of animals producing sperm. At the most effective hCG dose (300 IU), 100% of the male toads produced sperm, compared to only 35% for the best LHRH dose tested (4.0 micrograms). In addition to having a greater number of responders (P < 0.05), the 300 IU hCG treatment group had a much higher average sperm concentration (P < 0.05) than the treatment group receiving 4.0 micrograms LHRH. In contrast, these two treatments did not result in significant differences in sperm motility or quality of forward progressive motility. However, more males went into amplexus when treated with LHRH vs. hCG (90% vs. 75%) by nine hours post-administration. CONCLUSION: There is a clear dichotomy between the two hormones' physiological responses on gamete production and stimulation of amplexus. Understanding how these two hormones influence physiology and reproductive behaviors in amphibians will have direct bearing on establishing similar breeding protocols for endangered species.

Fecal near infrared spectroscopy to discriminate physiological status in giant pandas.

Giant panda (Ailuropoda melanoleuca) monitoring and research often require accurate estimates of population size and density. However, obtaining these estimates has been challenging. Innovative technologies, such as fecal near infrared reflectance spectroscopy (FNIRS), may be used to differentiate between sex, age class, and reproductive status as has been shown for several other species. The objective of this study was to determine if FNIRS could be similarly used for giant panda physiological discriminations. Based on samples from captive animals in four U.S. zoos, FNIRS calibrations correctly identified 78% of samples from adult males, 81% from adult females, 85% from adults, 89% from juveniles, 75% from pregnant and 70% from non-pregnant females. However, diet had an impact on the success of the calibrations. When diet was controlled for plant part such that "leaf only" feces were evaluated, FNIRS calibrations correctly identified 93% of samples from adult males and 95% from adult females. These data show that FNIRS has the potential to differentiate between the sex, age class, and reproductive status in the giant panda and may be applicable for surveying wild populations.

Applied reproductive technologies and genetic resource banking for amphibian conservation.

As amphibian populations continue to decline, both government and non-government organisations are establishing captive assurance colonies to secure populations deemed at risk of extinction if left in the wild. For the most part, little is known about the nutritional ecology, reproductive biology or husbandry needs of the animals placed into captive breeding programs. Because of this lack of knowledge, conservation biologists are currently facing the difficult task of maintaining and reproducing these species. Academic and zoo scientists are beginning to examine different technologies for maintaining the genetic diversity of founder populations brought out of the wild before the animals become extinct from rapidly spreading epizootic diseases. One such technology is genetic resource banking and applied reproductive technologies for species that are difficult to reproduce reliably in captivity. Significant advances have been made in the last decade for amphibian assisted reproduction including the use of exogenous hormones for induction of spermiation and ovulation, in vitro fertilisation, short-term cold storage of gametes and long-term cryopreservation of spermatozoa. These scientific breakthroughs for a select few species will no doubt serve as models for future assisted breeding protocols and the increasing number of amphibians requiring conservation intervention. However, the development of specialised assisted breeding protocols that can be applied to many different families of amphibians will likely require species-specific modifications considering their wide range of reproductive modes. The purpose of this review is to summarise the current state of knowledge in the area of assisted reproduction technologies and gene banking for the conservation of amphibians.

Corticosteroid-induced suppression of in vitro lymphocyte proliferation in four captive rhinoceros species.

Captive African black rhinoceroses (Diceros bicornis) are unusually susceptible to several diseases not commonly observed in any of the other three rhinoceros species maintained in captivity. The potential role of corticosteroids (either endogenously produced or exogenously administered) in the development of these sometimes fatal diseases has been questioned. In this study, the suppressive effects of two therapeutic corticosteroids (dexamethasone and hydrocortisone) on in vitro lymphocyte proliferation was examined in four rhinoceros species, including the Sumatran rhinoceros (Dicerorhinus sumatrensis, n = 3), Indian rhinoceros (Rhinoceros unicornis, n = 4), African black rhinoceros (n = 10), and African white rhinoceros (Ceratotherium simum, n = 5). Three blood samples collected from each rhinoceros 1 mo to 1 yr apart provided replicates for the study. Both dexamethasone and hydrocortisone suppressed (P < 0.05) lymphocyte proliferation stimulated by B-cell mitogens (pokeweed and lipopolysaccharide) and T-cell mitogens (phytohemagglutinin and concanavalin A). Suppressive effects of the glucocorticoids differed (P < 0.05) depending on the mitogen used to stimulate the lymphocytes. Overall, dexamethasone was a more potent suppressor of cell proliferation when compared with hydrocortisone (P < 0.05). However, black rhinoceros cell proliferation in response to any of the four mitogens was never completely suppressed, even in cultures containing the highest steroid concentration tested (10(-3) M). The effect of the two corticosteroids differed slightly among the rhinoceros species and subspecies tested, but there was no evidence that eastern or southern black rhinoceros lymphocytes were more sensitive to the suppressive effects of corticosteroids than the other rhinoceros species.

Comparative studies of mitogen- and antigen-induced lymphocyte proliferation in four captive rhinoceros species.

Cellular immune function in four rhinoceros species was evaluated by way of in vitro lymphocyte proliferation responses to mitogenic and antigenic stimuli to establish normative data on white blood cell activity for each species and to identify species-specific differences that might help explain the predisposition of black rhinoceroses (Diceros bicornis) to disease. A cross section of the U.S. rhinoceros population encompassing all four captive species was sampled, including the Sumatran rhinoceros (Dicerorhinus sumatrensis) (n = 3); Indian rhinoceros (Rhinoceros unicornis) (n = 4); African black rhinoceros (n = 16); and African white rhinoceros (Ceratotherium simum) (n = 10). Of the four species evaluated, African black rhinoceroses exhibited the weakest (P < 0.05) lymphocyte proliferative responses to the mitogens: pokeweed (0.1 microg/ml), phytohemagglutinin (0.3 microg/ml), and concanavalin A (5.0 microg/ml). Total cell density at the end of culture was only 70% of that achieved with lymphocytes isolated from African white rhinoceroses, Indian rhinoceroses, and Sumatran rhinoceroses. However, lymphocyte response to bacterial endotoxin lipopolysaccharide was similar (P > 0.05) across species. Antigenic stimulation produced much weaker responses than mitogenic stimulation. No differences (P > 0.05) were observed among rhinoceros species in response to 1 and 10 microg/ml of Leptospira icterohemorrhagiae or Leptospira gryppotyphosa. Lymphocytes from African white rhinoceroses proliferated weakly in the presence of Aspergillus fumigatus filtrate, whereas lymphocytes from the southern black rhinoceros subspecies appeared slightly suppressed in the presence of increasing doses (0.1, 1, and 10 microg/ml) of Aspergillus filtrate. This comparative data set characterizing lymphocyte proliferation in the rhinoceros reveals several differences in immune cell responses among rhinoceros species and provides some evidence that lymphocytes of captive African black rhinoceroses are less vigorous than those of the other rhinoceros species.

Structural and functional aspects of Bufo americanus spermatozoa: effects of inactivation and reactivation.

Very little is known about the effects of manipulating toad sperm activity in vitro, and such information is important in the development of a genetic resource bank for bufonid species. The specific objectives of this study were to: 1). identify the optimal inactivation and reactivation solutions for toad spermatozoa collected in urine; 2). establish the length of time toad spermatozoa can be exposed to an inactivation buffer and still resume motility upon reactivation; 3). evaluate the consequence of inactivation on specific sperm characteristics; and 4). characterize the sperm mitochondria vesicle (MV) and its relationship to motility. Reactivated sperm motility was similar after inactivation in either Simplified Amphibian Ringers (SAR) solution or DeBoer's (DB) solution. Diluting the buffer by 80% with water provided the best method for reactivating sperm. Dilutions with NaCl solutions (10-50 mM) produced inferior results. SAR-inactivated spermatozoa could remain suspended up to 4 hr and still regain 25% of initial motility upon reactivation in water. Compared to the controls, sperm motility was greater (P<0.01) over time for samples treated with SAR, although forward progression was significantly lower. Furthermore, SAR treatment resulted in sperm samples with a greater number of viable, morphologically normal, and intact MVs over time. Electron microscopy and fluorescent staining confirmed that the toad sperm's MV contains a large number of active mitochondria with very few other cytoplasmic structures. Nearly all spermatozoa exhibiting motility had an intact MV, and dissociation of this structure was clearly related to motility loss. In conclusion, toad spermatozoa can be effectively inactivated and reactivated by varying the osmolality of the external solutions and, although sperm forward progression is reduced, all other characteristics are well maintained. Moreover, the increased number of spermatozoa with intact MV after inactivation suggests the process may help preserve this important structure.

Proton-coupled electron transfer in Fe-superoxide dismutase and Mn-superoxide dismutase.

Fe-containing superoxide dismutase (FeSOD) and MnSOD are widely assumed to employ the same catalytic mechanism. However this has not been completely tested. In 1985, Bull and Fee showed that FeSOD took up a proton upon reduction [J. Am. Chem. Soc. 107 (1985) 3295]. We now demonstrate that MnSOD incorporates the same crucial coupling between electron transfer and proton transfer. The redox-coupled H(+) acceptor has been presumed to be the coordinated solvent molecule, in both FeSOD and MnSOD, however this is very difficult to test experimentally. We have now examined the most plausible alternative: that Tyr34 accepts a proton upon SOD reduction. We report specific incorporation of 13C in the C(zeta) positions of Tyr residues, assignment of the C(zeta) signal of Tyr34 in each of oxidized FeSOD and MnSOD, and direct NMR observations showing that in both cases, Tyr34 is in the neutral protonated state. Thus Tyr34 cannot accept a proton upon SOD reduction, and coordinated solvent is concluded to be the redox-coupled H(+) acceptor instead, in both FeSOD and MnSOD. We have also confirmed by direct 13C observation that the pK of 8.5 of reduced FeSOD corresponds to deprotonation of Tyr34. This work thus provides experimental proof of important commonalities between the detailed mechanisms of FeSOD and MnSOD.