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Abstract Background The scientific literature contains many examples where DNA sequence analyses have been used to provide definitive answers to phylogenetic problems that traditional (non-DNA based) approaches alone have failed to resolve. One notable example concerns the rhinoceroses, a group for which several contradictory phylogenies were proposed on the basis of morphology, then apparently resolved using mitochondrial DNA fragments. Results In this study we report the first complete mitochondrial genome sequences of the extinct ice-age woolly rhinoceros (Coelodonta antiquitatis), and the threatened Javan (Rhinoceros sondaicus), Sumatran (Dicerorhinus sumatrensis), and black (Diceros bicornis) rhinoceroses. In combination with the previously published mitochondrial genomes of the white (Ceratotherium simum) and Indian (Rhinoceros unicornis) rhinoceroses, this data set putatively enables reconstruction of the rhinoceros phylogeny. While the six species cluster into three strongly supported sister-pairings: (i) The black/white, (ii) the woolly/Sumatran, and (iii) the Javan/Indian, resolution of the higher-level relationships has no statistical support. The phylogenetic signal from individual genes is highly diffuse, with mixed topological support from different genes. Furthermore, the choice of outgroup (horse vs tapir) has considerable effect on reconstruction of the phylogeny. The lack of resolution is suggestive of a hard polytomy at the base of crown-group Rhinocerotidae, and this is supported by an investigation of the relative branch lengths. Conclusion Satisfactory resolution of the rhinoceros phylogeny may not be achievable without additional analyses of substantial amounts of nuclear DNA. This study provides a compelling demonstration that, in spite of substantial sequence length, there are significant limitations with single-locus phylogenetics. We expect further examples of this to appear as next-generation, large-scale sequencing of complete mitochondrial genomes becomes commonplace in evolutionary studies. "The human factor in classification is nowhere more evident than in dealing with this superfamily (Rhinocerotoidea)." G. G. Simpson (1945)

Nonbreeding white rhino bulls are traditionally housed individually in zoos, but space limitations require the exploration of alternative techniques. There are no studies of group-housed bulls, and the feasibility of managing a bachelor group is unknown. Six bulls (two bulls each of 3-, 5-, and 8-yr-olds) in a 3.1 ha enclosure were observed for 30 min each 2×/wk for 20–31 wks. The 3-yr-olds were relocated after the first 20 weeks. Rhino location, nearest neighbor, aggressive behaviors, and dominance interactions were recorded. There was no evidence of habitat spatial segregation. The 3- and 5-yr-olds grouped together by age; 8-yr-olds did not associate as much as younger rhinos. Aggressive vocalizations and physical aggression were infrequent (range 0–1.5/h/bull and 0–1/h/bull, respectively). Eight-year-olds displayed and received aggressive vocalizations the most; three-year-olds displayed and received physical aggression the most. After the 3-yr-olds were removed, the habitat usage and social proximity of the remaining bulls did not change. Aggressive behaviors increased, but vocalizations (range 0.18–3.1/h/bull) remained more frequent than physical aggression (range 0.18–0.73/h/bull). The highest-ranking 8-yr-old engaged in physical altercations the least. The bulls shared space, and the use of vocalizations more than physical aggression deterred injury. These findings indicate that bachelor group management is feasible in a large enclosure with young bulls. [ABSTRACT FROM AUTHOR]

More than 50% of caudates are threatened with extinction and are in need of ex-situ breeding programs to support conservation efforts and species recovery. Unfortunately, many salamander populations under human care can experience reproductive failure, primarily due to missing environmental cues necessary for breeding. Assisted reproductive technologies (ARTs) are a useful suite of techniques for overcoming or bypassing these missing environmental cues to promote breeding. Exogenous hormones are used to stimulate natural breeding behaviors or gamete expression for in-vitro fertilization or biobanking and are typically administered intramuscularly in caudates. While effective, intramuscular injection is risky to perform in smaller-bodied animals, resulting in health and welfare risks. This research investigated the spermiation response to hormone administration through a non-invasive oral bioencapsulation route using the tiger salamander (Ambystoma tigrinum) as a model species. Male salamanders were randomly rotated six weeks apart through four treatments (n = 11 males/treatment) in which animals received a resolving dose of gonadotropin-releasing hormone (GnRH) as follows: (1) Prime-Only (0.0 μg/g); (2) Low (0.25 μg/g); (3) Medium (1.0 μg/g); and (4) High (2.0 μg/g). All males were given a GnRH priming dose (0.25 μg/g) 24 hours prior to the resolving dose. Exogenous hormone was delivered inside of a cricket (Gryllodes sigillatus) that was presented as a food item by tweezers. Sperm samples were collected at 1, 3, 6, 9, 12, and 24 hours after the resolving dose and analyzed for quantity and quality. For all treatments, sperm concentration was produced in an episodic pattern over time. The Prime-Only treatment had a lower (p < 0.05) percent of sperm exhibiting normal morphology compared to treatments utilizing a resolving dose of GnRH. Overall, oral administration of GnRH is a feasible route of inducing spermiation in salamanders, yielding sperm of sufficient quantity and quality for in-vitro fertilization and biobanking efforts. [ABSTRACT FROM AUTHOR]

Reliable pregnancy diagnostics would be beneficial for monitoring polar bear (Ursus maritimus) populations both in situ and ex situ, but currently there is no method of non-invasive pregnancy detection in this species. Recent reports in several carnivore species described the identification of fecal proteins that may serve as pregnancy biomarkers; however, repeatability has been limited. The objective of the current analysis was to utilize an unbiased, antibody-free, label-free method for the identification and quantification of fecal proteins to determine if differences associated with pregnancy are detectable in polar bears. Protein was extracted from fecal samples (n = 48) obtained from parturient (n = 6) and non-parturient (n = 6) profiles each at four timepoints: pre-breeding season, embryonic diapause, early placental pregnancy, and mid-placental pregnancy. Protein was prepared and analyzed on the Thermo Orbitrap Eclipse nanoLC-MS/MS system. A total of 312 proteins was identified and quantified; however, coefficients of variation (CV) were high for both abundance ratio variability (384.8 ± 61.0% SEM) and within group variability (86.8 ± 1.5%). Results of this study suggest that the inconsistencies in specific protein concentrations revealed previously by antibody-based assays may not be due to that methodology's limitations, but rather, are reflective of true variation that exists among samples. [ABSTRACT FROM AUTHOR]

A consequence of the poaching crisis is that managed rhinoceros populations are increasingly important for species conservation. However, black rhinoceroses (BR; Diceros bicornis) and Sumatran rhinoceroses (SR; Dicerorhinus Sumatrensis) in human care often store excessive iron in organ tissues, a condition termed iron overload disorder (IOD). IOD research is impeded by the challenge of accurately monitoring body iron load in living rhinoceroses. The goals of this study were to (i) determine if labile plasma iron (LPI) is an accurate IOD biomarker and (ii) identify factors associated with iron-independent serum oxidative reduction potential (ORP). Serum (106 samples) from SRs (n  = 8), BRs (n  = 28), white rhinoceros (n  = 24) and greater one-horned rhinoceros (GOH; n  = 16) was analysed for LPI. Samples from all four species tested positive for LPI, and a higher proportion of GOH rhinoceros samples were LPI positive compared with those of the other three species (P  < 0.05). In SRs, the only LPI-positive samples were those from individuals clinically ill with IOD, but samples from outwardly healthy individuals of the other three species were LPI positive. Serum ORP was lower in SRs compared with that in the other three species (P  < 0.001), and iron chelation only reduced ORP in the GOH species (P  < 0.01; ~5%). Serum ORP sex bias was revealed in three species with males exhibiting higher ORP than females (P  < 0.001), the exception being the SR in which ORP was low for both sexes. ORP was not associated with age or serum iron concentrations (P  ≥ 0.05), but was positively correlated with ferritin (P  < 0.01). The disconnect between LPI and IOD was unanticipated, and LPI cannot be recommended as a biomarker of advanced rhino IOD. However, data provide valuable insight into the complex puzzle of rhinoceros IOD. [ABSTRACT FROM AUTHOR]

Iron overload disorder (IOD) affects many wildlife species cared for ex situ. Two of the four rhinoceros species in human care, Sumatran rhinoceros (Dicerorhinus sumatrensis) and black rhinoceros (Diceros bicornis), are susceptible, whereas the other two, white rhinoceros (Ceratotherium simum) and greater one-horned (GOH) rhinoceros (Rhinoceros unicornis), are relatively resistant to IOD. Complex interrelationships exist between mammalian hosts, their indigenous gut microbiota, metabolome, physical condition, and iron availability. The goal of this study was to gain insight into these relationships within the family Rhinocerotidae. Specific objectives were to (1) characterize the gut microbiome and metabolome of four rhinoceros species; (2) compare the microbiome and metabolome of IOD-susceptible and IOD-resistant rhinoceros species; and (3) identify variation in the microbiome and metabolome associated with compromised health or disease in IOD-susceptible rhinoceroses. Fecal samples were collected from 31 rhinoceroses (Sumatran rhinoceros, n = 3; black rhinoceros, n = 6; GOH rhinoceros, n = 9; white rhinoceros, n = 13) located at five facilities, and matched fecal aliquots were processed for microbiome and metabolome analyses using 16S rRNA gene sequencing and nuclear magnetic resonance spectroscopy, respectively. Despite the phylogenetic disparity and dissimilar zoo diets of the hosts, the structure of the fecal microbiota of the two IOD-susceptible rhinoceros species were more closely related to each other than to those of the two IOD-resistant species (Bray–Curtis dissimilarity; IOD-susceptible vs. IOD-resistant p -value < 0.001). In addition, IOD-susceptible rhinoceroses exhibited less microbial diversity than their IOD-resistant relatives (Shannon diversity; p -value < 0.001) which could have health implications. Of note, the black rhinoceros was distinct among the four rhinoceros species with the most divergent fecal metabolome; interestingly, it contained higher concentrations of short chain fatty acids. Neither age nor sex were associated with differences in microbial community composition (p = 0.253 and 0.488, respectively) or fecal metabolomic profile (p = 0.634 and 0.332, respectively). Differences in the distal gut microbiomes between IOD-resistant and IOD-susceptible rhinoceroses support hypotheses that gut microbes play a role in host iron acquisition, and further studies and experiments to test these hypotheses are warranted. [ABSTRACT FROM AUTHOR]

The aim of this study was to determine if ferritin is a reliable biomarker of iron overload disorder (IOD) progression and hemochromatosis in the Sumatran rhinoceros ( Dicerorhinus sumatrensis) by developing a species-specific ferritin assay and testing historically banked samples collected from rhinos that did and did not die of hemochromatosis. Ferritin extracted from Sumatran rhino liver tissue was used to generate antibodies for the Enzyme Immunoassay. Historically banked Sumatran rhino serum samples ( n = 298) obtained from six rhinos in US zoos ( n = 290); five rhinos at the Sumatran Rhino Conservation Centre in Sungai Dusun, Malaysia ( n = 5); and two rhinos in Sabah, Malaysia ( n = 3) were analyzed for ferritin concentrations. Across all US zoo samples, serum ferritin concentrations ranged from 348 to 7,071 ng/ml, with individual means ranging from 1,267 ( n = 25) to 2,604 ng/ml ( n = 36). The ferritin profiles were dynamic, and all rhinos exhibited spikes in ferritin above baseline during the sampling period. The rhino with the highest mean ferritin concentration did not die of hemochromatosis and exhibited only mild hemosiderosis postmortem. A reproductive female exhibited decreases and increases in serum ferritin concurrent with pregnant and nonpregnant states, respectively. Mean (±SD) serum ferritin concentration for Sumatran rhinos in Malaysia was high (4,904 ± 4,828 ng/ml) compared to that for US zoo rhinos (1,835 ± 495 ng/ml). However, those in Sabah had lower ferritin concentrations (1,025 ± 52.7 ng/ml) compared to those in Sungai Dusun (6,456 ± 4,941 ng/ml). In conclusion, Sumatran rhino serum ferritin concentrations are dynamic, and increases often are not associated with illness or hemochromatosis. Neither a specific pattern nor the individual's overall mean ferritin concentration can be used to accurately assess IOD progression or diagnose hemochromatosis in this rhino species. [ABSTRACT FROM AUTHOR]

Red pandas ( Ailurus fulgens styani) exhibit a variable gestation length and may experience a pseudopregnancy indistinguishable from true pregnancy; therefore, it is not possible to deduce an individual's true pregnancy status and parturition date based on breeding dates or fecal progesterone excretion patterns alone. The goal of this study was to evaluate the use of transabdominal ultrasonography for pregnancy diagnosis in red pandas. Two to three females were monitored over 4 consecutive years, generating a total of seven profiles (four pregnancies, two pseudopregnancies, and one lost pregnancy). Fecal samples were collected and assayed for progesterone (P4) and estrogen conjugate (EC) to characterize patterns associated with breeding activity and parturition events. Animals were trained for voluntary transabdominal ultrasound and examinations were performed weekly. Breeding behaviors and fecal EC data suggest that the estrus cycle of this species is 11-12 days in length. Fecal steroid metabolite analyses also revealed that neither P4 nor EC concentrations were suitable indicators of pregnancy in this species; however, a secondary increase in P4 occurred 69-71 days prior to parturition in all pregnant females, presumably coinciding with embryo implantation. Using ultrasonography, embryos were detected as early as 62 days post-breeding/50 days pre-partum and serial measurements of uterine lumen diameter were documented throughout four pregnancies. Advances in reproductive diagnostics, such as the implementation of ultrasonography, may facilitate improved husbandry of pregnant females and allow for the accurate prediction of parturition. [ABSTRACT FROM AUTHOR]

A variety of wildlife species maintained in captivity are susceptible to iron storage disease (ISD), or hemochromatosis, a disease resulting from the deposition of excess iron into insoluble iron clusters in soft tissue. Sumatran rhinoceros (Dicerorhinus sumatrensis) is one of the rhinoceros species that has evolutionarily adapted to a low-iron diet and is susceptible to iron overload. Hemosiderosis is reported at necropsy in many African black and Sumatran rhinoceroses but only a small number of animals reportedly die from hemochromatosis. The underlying cause and reasons for differences in susceptibility to hemochromatosis within the taxon remains unclear. Although serum ferritin concentrations have been useful in monitoring the progression of ISD in many species, there is some question regarding their value in diagnosing hemochromatosis in the Sumatran rhino. To investigate the metabolic changes during the development of hemochromatosis and possibly increase our understanding of its progression and individual susceptibility differences, the serum metabolome from a Sumatran rhinoceros was investigated by nuclear magnetic resonance (NMR)-based metabolomics. The study involved samples from female rhinoceros at the Cincinnati Zoo (n = 3), including two animals that died from liver failure caused by ISD, and the Sungai Dusun Rhinoceros Conservation Centre in Peninsular Malaysia (n = 4). Principal component analysis was performed to visually and statistically compare the metabolic profiles of the healthy animals. The results indicated that significant differences were present between the animals at the zoo and the animals in the conservation center. A comparison of the 43 serum metabolomes of three zoo rhinoceros showed two distinct groupings, healthy (n = 30) and unhealthy (n = 13). A total of eighteen altered metabolites were identified in healthy versus unhealthy samples. Results strongly suggest that NMR-based metabolomics is a valuable tool for animal health monitoring and may provide insight into the progression of this and other insidious diseases. [ABSTRACT FROM AUTHOR]

Urinary hormone analysis was conducted on two adult female Indian rhinoceroses ( Rhinoceros unicornis) that exhibited minimal or no estrual behaviors traditionally used to time breeding. Urine was collected throughout two consecutive estrous cycles to establish preliminary data on each individual's pattern and concentration of estrogen conjugates (EC) and progesterone metabolites (PdG) during follicular and luteal phases. Following preliminary endocrine analysis, urine samples were shipped on a frequent basis to verify when each female was off baseline in EC. Estrus and breeding dates were then predicted. Females were introduced to fresh male rhinoceros fecal samples daily throughout the follicular phase to potentially stimulate estrous behaviors. Despite successful assessment of follicular phase dynamics, females sometimes failed to exhibit estrus. Both females conceived following mating introductions that were timed using hormone analysis. Pregnancy was diagnosed either by endocrine analysis or rectal ultrasonography. Progestational support (altrenogest) occurred after pregnancy confirmation and varied for each female (21 and 66 days post-breeding). One female experienced early pregnancy loss and the other successfully completed a term pregnancy. These results demonstrate that a science based management strategy that relies on urinary biomarkers of ovarian function can facilitate naturally breeding captive Indian rhinoceroses. Zoo Biol. 33:83-88, 2014. © 2013 Wiley Periodicals Inc. [ABSTRACT FROM AUTHOR]

To help save the Sumatran rhino from extinction, the captive breeding program must capitalize on each rhino's reproductive lifespan. Doing so requires knowing when calves are sexually mature. The goal of this study was to monitor physiological changes associated with sexual maturation in two captive born calves (one male and one female) to determine the approximate age of maturity for both sexes of this species. Fecal testosterone metabolites were monitored in the male calf from 6 months to 7 years of age, and fecal pregnane metabolites were measured in the female calf from 6 months to 5.5 years of age. In addition, rectal ultrasonography was employed to monitor changes in ovarian activity from 2 to 5.5 years of age. The male calf's fecal testosterone concentrations reached levels comparable to those detected in samples from adult males when he was 6-6.5 years of age. The first pre-ovulatory sized follicle was observed on the ovaries of the female calf when she was 4.75 years old, but fecal pregnane metabolite concentrations only reached maximum mean concentrations and variability when she was 5-5.5 years of age. Results from this study indicate that male and female Sumatran rhino calves are sexually mature at 6-6.5 and 5-5.5 years of age, respectively. Zoo Biol. 32:549-555, 2013. © 2013 Wiley Periodicals Inc. [ABSTRACT FROM AUTHOR]

Simple Summary: Polar bears are threatened by habitat loss, decreased food availability, and reduced reproductive success due to climate change. Zoo populations can support species survival through preservation of genetic diversity and maintenance of insurance populations, but in the US, the zoo polar bear population is currently not sustainable. The development of sperm collection and cryopreservation can help to support the population by providing the biomaterial needed for assisted reproductive techniques, such as artificial insemination. However, these procedures are not well described for polar bears. Data from 38 opportunistic sperm collections, that were conducted between 2011 and 2021, were assessed to establish best practices to date for collecting and preserving polar bear sperm. The information gathered demonstrates that urethral catheterization is an efficient method of sperm collection, sperm can be rescued postmortem from the vasa deferentia and epididymides, and polar bear sperm collection appears to be most effective during the breeding season. Furthermore, polar bear sperm can survive cryopreservation. Further studies will optimize these techniques, but this summary provides information that is immediately applicable to enhancing sample collection and cryopreservation success that could support the long-term genetic management of polar bears in zoos. Assisted reproductive technologies can aid conservation efforts via support of ex situ population management and preservation of genetic material. Data from 38 sperm collection attempts from 17 polar bears (1–5 procedures/bear) were evaluated. Sample collections were attempted via electroejaculation (EEJ; n = 6), urethral catheterization (UC; n = 25), or sperm rescue (SR; n = 7) during the breeding season (Jan. 1-May 21; n = 27) and nonbreeding season (May 22-Dec. 31; n = 11). Sperm retrieval was successful in 1 EEJ (16.7%), 18 UC (72.0%) and 4 SR (57.1%) collections. Initial sperm motility and viability were 50.0% and 77.0% for EEJ, 64.3 ± 7.4% and 80.9 ± 3.8% for UC, and 56.7 ± 8.8% and 80.5 ± 0.5% for SR. UC and SR were more likely to be successful during the breeding season (84.2–100%) than the nonbreeding season (25.0–33.3%). Testicular tumors were observed in four males (57%) during SR. In total, 13 samples were cryopreserved (n = 1 EEJ, 9 UC, and 3 SR) with egg-yolk-based equine extender (EQ) or OptiXcell (OP). For both extenders, post-thaw motility and viability were reduced by 20–60% and 30–65%, respectively. Further efforts to optimize procedures are warranted, but this summary provides data useful for enhancing the success of polar bear sperm collection and cryopreservation. [ABSTRACT FROM AUTHOR]

Background: The scientific literature contains many examples where DNA sequence analyses have been used to provide definitive answers to phylogenetic problems that traditional (non-DNA based) approaches alone have failed to resolve. One notable example concerns the rhinoceroses, a group for which several contradictory phylogenies were proposed on the basis of morphology, then apparently resolved using mitochondrial DNA fragments. Results: In this study we report the first complete mitochondrial genome sequences of the extinct ice-age woolly rhinoceros (Coelodonta antiquitatis), and the threatened Javan (Rhinoceros sondaicus), Sumatran (Dicerorhinus sumatrensis), and black (Diceros bicornis) rhinoceroses. In combination with the previously published mitochondrial genomes of the white (Ceratotherium simum) and Indian (Rhinoceros unicornis) rhinoceroses, this data set putatively enables reconstruction of the rhinoceros phylogeny. While the six species cluster into three strongly supported sister-pairings: (i) The black/white, (ii) the woolly/Sumatran, and (iii) the Javan/Indian, resolution of the higher-level relationships has no statistical support. The phylogenetic signal from individual genes is highly diffuse, with mixed topological support from different genes. Furthermore, the choice of outgroup (horse vs tapir) has considerable effect on reconstruction of the phylogeny. The lack of resolution is suggestive of a hard polytomy at the base of crown-group Rhinocerotidae, and this is supported by an investigation of the relative branch lengths. Conclusion: Satisfactory resolution of the rhinoceros phylogeny may not be achievable without additional analyses of substantial amounts of nuclear DNA. This study provides a compelling demonstration that, in spite of substantial sequence length, there are significant limitations with single-locus phylogenetics. We expect further examples of this to appear as next-generation, large-scale sequencing of complete mitochondrial genomes becomes commonplace in evolutionary studies. "The human factor in classification is nowhere more evident than in dealing with this superfamily (Rhinocerotoidea)." G. G. Simpson (1945) [ABSTRACT FROM AUTHOR]

Investigates the follicular, endocrine and behavioral dynamics of the Indian rhinoceros oestrous cycle. Administration of longitudinal evaluations of in two young Indian female rhinoceroses to characterize the oestrous cycle; Occurrence of ovulation in all cycles; Confirmation of follicular collapse following the onset of behavioral oestrous in ovulatory cycles.

The specific objectives of the present study were to investigate the effects of manual seeding, differing freeze and thaw rates as well as storage for 24 h at 4 °C prior to cryopreservation on post-thaw sperm quality in Asian elephants. Extended semen was cooled in an equitainer to 4 °C, frozen in liquid nitrogen vapour at various rates with and without manual seeding or in a dry shipper and thawed at 37, 50 and 75 °C. There was a significant effect of freeze rate on post-thaw motility (P < 0.0001) and acrosomal integrity (P < 0.005). The faster freeze rates in the dry shipper and at 1 cm or 2 cm above liquid nitrogen consistently provided better cryopreservation than slower freezing rates. Thaw temperature had no effect on post-thaw semen quality but there was an interaction between freeze and thaw rates with higher thaw rates resulting in superior post-thaw semen quality in straws frozen at fast rates. Storage of samples prior to freezing had a detrimental effect on post-thaw semen quality. In summary, our results indicate cooling extended semen in an equitainer and cryopreserving it by placing straws directly in a dry shipper is a simple technique for effectively cryopreserving Asian elephant semen in the field or zoo. [ABSTRACT FROM AUTHOR]

Validation of androgen enzyme immunoassays (EIA) using purified antiserum with a high cross-reactivity for testosterone and 5α-dihydrotestosterone in polar bear urine (PBU) has previously been reported. However, the cross-reactivity of this antiserum with urinary testosterone metabolites was not determined. Therefore, our objective was to perform high performance liquid chromatography (HPLC) analysis of PBU to describe the major immunoreactive androgens and metabolites present, and to determine if enzy-matic hydrolysis (EH) of PBU prior to EIA would improve our detection of androgen secretion during different reproductive states. EH of urinary conjugated steroids was performed using ß-glucuroni¬dase-arylsulfatase. Three buffer treatments were tested to determine if buffer type and/or pH negatively influenced EIA parameters. Due to lower matrix interference, PBS (pH 5.0) was selected for subsequent EH. Androgen concentrations of neat and hydrolyzed PBU assayed directly and after extraction were highly correlated (r ≥ 0.954, p < 0.012). HPLC supported these findings, whereby the fraction of neat urine eluting at the same time as the known testosterone-glucuronide standard demonstrated high immunoreactivity after, but not before, EH. Profiles of a breeding and a non-breeding female using parallel fecal, neat PBU, and hydrolyzed PBU samples displayed comparable patterns of androgen secretion, but discrete changes in androgen concentrations were better detected using the latter. These results indicate that accurate analysis of urinary androgen concentrations in the polar bear can be achieved following EH without an extraction step, thus saving substantial time which may critically influence the success of natural or assisted breeding management decisions. [ABSTRACT FROM AUTHOR]

The objective of this study was to design an artificial insemination (AI) protocol using cryopreserved spermatozoa to obtain pregnancies in captive Indian rhinoceroses ( Rhinoceros unicornis ). Four methods developed varied by timing and approach, as follows; Method 1: females ( n = 2) were inseminated pre- and post-ovulation under general anesthesia, Method 2: females ( n = 2) were inseminated pre-ovulation without anesthetic via endoscopy, Method 3: females ( n = 1) were inseminated pre-ovulation without anesthetic via manual insertion of an insemination catheter, Method 4: females ( n = 2) were inseminated same as Method 3 with the addition of standing sedation. Semen deposition site varied as a result of changes in AI technology and experience. All females conceived following intrauterine AI using three methods. Four pregnancies ( n = 3 females) produced via Method 3 and 4 resulted in term births ( n = 2 male calves, n = 2 female calves) at 481.8 ± 12.8 days post-AI. Unfortunately, two early pregnancy losses were documented in a fourth female conceiving via Method 2. Pregnancy rates were 0%, 22%, 17%, and 50% for Method 1–4, respectively. Method 3 and 4 rates improved to 29% and 67%, respectively when accounting for AI’s conducted only on ovulatory estrous cycles. Spermatozoa ( n = 5 males) were cryopreserved 0.3–9.3 y prior to successful AI procedures. The lowest dose of frozen-thawed sperm resulting in conception was 500 × 10 6 motile sperm. Mean time from AI to ovulation in conceptive and non-conceptive cycles was 26 ± 11.8 h and 66 ± 80.7 h, respectively. [ABSTRACT FROM AUTHOR]

Despite serum ferritin's potential as an iron status indicator, its concentrations vary significantly throughout a black rhinoceros's (Diceros bicornis) life, sometimes irrespective of iron load. We explored acute phase response-related biomarkers, serum amyloid A (SAA) and ceruloplasmin (Cp), to better understand the mechanisms influencing serum ferritin changes in managed black rhinoceroses. The objective was to evaluate the relationships between circulating levels of ferritin, SAA, and Cp in black and white rhinoceroses (Ceratotherium simum). We analyzed banked serum samples collected serially from 11 black (n = 222) and 7 white (n = 134), rhinoceroses (rhinos) and classified samples based on SAA values: clinically healthy (< 1 mg/L), subclinical (1-7 mg/L), or clinically abnormal (> 7 mg/L). In black rhinos, serum ferritin was not different between health status categories (p = 0.5292), nor was it correlated with SAA (p = 0.4164). However, Cp activity was significantly lower in clinically healthy sera (p < 0.0001) and had a moderate positive association with SAA (r = 0.477, p < 0.0001). Among the white rhino samples, only five had SAA values greater than 1 mg/L, limiting the assessment of ferritin and Cp activity in the health context. Minor, yet significant, relationships were observed between serum ferritin and Cp activity, negative in black rhinos (r = -0.206; p = 0.0022) and positive in white rhinos (r = 0.289, p = 0.0008). Cp activity may aid in diagnosing illness in black and white rhinos, based on values observed in sera collected near the time of death. However, acute inflammatory processes do not appear to be one of the primary drivers of the high ferritin concentrations detected in some black rhinos., (© 2024 The Author(s). Zoo Biology published by Wiley Periodicals LLC.)

Iron overload disorder (IOD) in critically endangered Sumatran ( Dicerorhinus sumatrensis ) and black ( Diceros bicornis ) rhinoceros is an over-accumulation of iron in organs which may exacerbate other diseases and indicate metabolic disturbances. IOD in rhinos is not well understood and diagnostics and therapeutics are limited in effectiveness. MicroRNAs (miRNAs) are small non-coding RNAs capable of altering protein synthesis. miRNA expression responds to physiological states and could serve as the basis for development of diagnostics and therapeutics. This study aimed to identify miRNAs differentially expressed among healthy rhinos and those afflicted with IOD or other diseases ("unhealthy"), and assess expression of select miRNAs to evaluate their potential as biomarkers of IOD. miRNAs in serum of black ( n = 11 samples; five individuals) and Sumatran ( n = 7 samples; four individuals) rhinos, representing individuals categorized as healthy ( n = 9), unhealthy ( n = 5), and afflicted by IOD ( n = 3) were sequenced. In total, 715 miRNAs were identified, of which 160 were novel, 131 were specific to black rhinos, and 108 were specific to Sumatran rhinos. Additionally, 95 miRNAs were specific to healthy individuals, 31 specific to unhealthy, and 63 were specific to IOD individuals. Among healthy, unhealthy, and IOD states, 21 miRNAs were differentially expressed ( P ≤ 0.01). Five known miRNAs (let-7g, miR-16b, miR-30e, miR-143, and miR-146a) were selected for further assessment via RT-qPCR in serum from black ( n = 61 samples; seven individuals) and Sumatran ( n = 38 samples; five individuals) rhinos. let-7g, miR-30e, and miR-143 all showed significant increased expression ( P ≤ 0.05) during IOD (between 1 and 2 years prior to death) and late IOD (within 1 year of death) compared to healthy and unhealthy individuals. miR-16b expression increased ( P ≤ 0.05) in late IOD, but was not different among IOD, healthy, and unhealthy states ( P > 0.05). Expression of miR-146a increased in IOD and late IOD as compared to unhealthy samples ( P ≤ 0.05) but was not different from the healthy state ( P > 0.05). Selected serum miRNAs of black and Sumatran rhinos, in particular let-7g, miR-30e, and miR-143, could therefore provide a tool for advancing rhino IOD diagnostics that should be further investigated., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Wojtusik, Curry and Roth.)

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., (Copyright 2003 Wiley-Liss, Inc.)