Label-Free Quantification (LFQ) of Fecal Proteins for Potential Pregnancy Detection in Polar Bears.

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.

Case Studies in Polar Bear (Ursus maritimus) Sperm Collection and Cryopreservation Techniques.

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.

Rhinoceros serum labile plasma iron and associated redox potential: interspecific variation, sex bias and iron overload disorder disconnect.

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.

Rhinoceros Serum microRNAs: Identification, Characterization, and Evaluation of Potential Iron Overload Biomarkers.

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.

Animal protein-free OptiXcell and shortened equilibration periods can replace egg yolk-based extender and slow cooling for rhinoceros semen cryopreservation.

OptiXcell (OP) was tested as an animal protein-free alternative to an egg yolk-based extender for rhinoceros semen cryopreservation and shorter chilling/equilibration periods were evaluated. Semen was collected from three rhinoceros species: black (Diceros bicornis; n = 2), white (Ceratotherium simum; n = 2), and greater one-horned (GOH; Rhinoceros unicornis; n = 3). Controls were diluted with equine extender (EQ) or OP and equilibrated for 1 h. Treatments were diluted with extender and cooled for 15 min (fast: FEQ; FOP) or not cooled (immediate: IEQ; IOP), prior to cryopreservation. Motility decreased post-thaw (EQ: 50.7 ±â€¯5.2%; OP: 52.9 ±â€¯3.4%) from fresh (82.9 ±â€¯2.9%), was higher in OP than IOP (38.6 ±â€¯4.9%; P ≤ 0.05) and decreased over time (P ≤ 0.05). Post-thaw acrosomal integrity was lower in EQ, FEQ, and IEQ (56.9 ±â€¯0.7; 56.6 ±â€¯4.5; 54.9 ±â€¯2.9%) than OP, FOP, IOP (71.8 ±â€¯4.7; 71.9 ±â€¯3.8; 69.9 ±â€¯4.5%) and fresh (72.6 ±â€¯1.4%; P ≤ 0.05). Progression and viability were lower in EQ (2.8 ±â€¯0.2; 61.9 ±â€¯7.4%) and OP (3.1 ±â€¯0.2; 53.4 ±â€¯6.9%) than fresh (3.7 ±â€¯0.2; 87.2 ±â€¯1.3%), decreased over time (P ≤ 0.05) but not different among treatments (P > 0.05). Morphology did not differ between fresh (75.0 ±â€¯4.9% normal) and any treatment group (70.0-77.8%) or over time (P > 0.05). OptiXcell is comparable to egg yolk-based EQ when used for rhinoceros semen cryopreservation. Furthermore, chilling/equilibration can be reduced with little impact on sperm characteristics.

Comparison of soy lecithin, coconut water, and coconut milk as substitutes for egg-yolk in semen cryodiluent for black rhinoceros (Diceros bicornis) and Indian rhinoceros (Rhinoceros unicornis).

Semen cryopreservation for the black rhinoceros (Diceros bicornis) and Indian rhinoceros (Rhinoceros unicornis) relies on extenders containing egg-yolk (EY). Use of such media is not ideal as inter-batch composition varies and there is risk of pathogenic contamination. The goal of this study was to test animal protein-free extenders. Semen collected via electroejaculation from 10 rhinoceros (6 black, 4 Indian) was diluted with extender containing EY, 1% or 2% soy lecithin (1%SL; 2%SL), coconut water (CW), or coconut milk (CM), cryopreserved and evaluated for sperm motility, viability, morphology, progression, and acrosomal integrity at 0, 1, 3, 6 and 24 h post-thaw. Mean ±â€¯SD fresh ejaculate motility was 84.5 ±â€¯7.6%, progression: 3.6 ±â€¯0.6 (scale 0-5), viability: 83.4 ±â€¯7.1%, intact acrosomes: 71.3 ±â€¯6.9%, and morphologically normal: 78.8 ±â€¯13.6%. Motility and progression decreased in all groups post-thaw, were greatest in EY, and decreased over time (P ≤ 0.05). Motility and progression did not differ (P > 0.05) between 1%SL and 2%SL, but were lower (P ≤ 0.05) in CM and CW, and acrosomal integrity was higher (P ≤ 0.05) in EY, 1%SL and 2%SL than in CM and CW. Post-thaw viability was greatest in EY and 2%SL followed by 1%SL, then CM and CW (P ≤ 0.05). Morphology did not differ among treatments (P > 0.05). Morphology, acrosomal integrity, and viability were maintained over time (P > 0.05). Although some rhinoceros sperm survived cryopreservation in SL treatments, reduced post-thaw motility rendered all treatments inadequate substitutes for EY-based extenders.

INVESTIGATION OF FACTORS POTENTIALLY ASSOCIATED WITH SERUM FERRITIN CONCENTRATIONS IN THE BLACK RHINOCEROS ( DICEROS BICORNIS) USING A VALIDATED RHINOCEROS-SPECIFIC ASSAY.

Iron overload disorder (IOD) can lead to organ dysfunction and may exacerbate other diseases in the critically endangered black rhinoceros ( Diceros bicornis). It is important to develop methods for monitoring the progression of iron storage (hemosiderosis), diagnosing the disease, and evaluating treatments in this species. Traditionally, an equine enzyme immunoassay (EIA) was used to measure rhinoceros ferritin, a serum protein correlated to iron stores. The goal of this study was to validate a rhinoceros-specific assay and investigate factors potentially associated with ferritin concentrations in black rhinoceros. A ferritin EIA developed for Sumatran rhinoceros was validated for black rhinoceros via Western blot analysis of liver ferritin and confirmed parallelism of serum samples to the EIA standard curve and used to analyze serum samples ( n = 943) collected from 36 black rhinoceros (<1-33 yr) at 14 U.S. institutions. Mean (±SEM) serum ferritin concentration was 6,738 ± 518 ng/ml (range: 85-168,451 ng/ml). Concentrations differed among individuals with eastern black rhinoceros (7,444 ± 1,130 ng/ml) having a higher mean ferritin than southern black rhinoceros (6,317 ± 505 ng/ml; P < 0.05) and higher mean values in wild-born (11,110 ± 1,111 ng/ml) than captive-born individuals (3,487 ± 293 ng/ml; P < 0.05). Ferritin concentrations did not differ between young rhinoceros (<5 yr old; 2,163 ± 254 ng/ml) and adults (7,623 ± 610 ng/ml) and were not correlated with age ( r = 0.143) or time in captivity ( r = 0.146, wild born; r = 0.104, all animals). Ferritin concentration was not impacted by sex (female: 2,086 ± 190 ng/ml; male: 8,684 ± 717 ng/ml), date, month, or season of collection ( P > 0.05). Data indicate ferritin concentrations are variable and not necessarily associated with IOD; ferritin is not recommended for diagnosing or monitoring IOD in black rhinoceros.

Pretreatment with cholesterol-loaded cyclodextrins prevents loss of motility associated proteins during cryopreservation of addra gazelle (Nanger dama ruficollis) spermatozoa.

Sperm cryopreservation is challenging, often resulting in irreversible damage to spermatozoa, as indicated by decreased motility, viability, and/or acrosomal integrity. Developing cryopreservation protocols for gametes of endangered species compounds the complexity of technique optimization; samples are difficult to obtain and numbers are limited. Cryopreservation of sperm collected from the critically endangered addra gazelle (Nanger dama ruficollis), a member of the Bovidae family, resulted in significant loss of motility, which was prevented by pretreatment with cholesterol-loaded cyclodextrin (CLC). This study investigated the proteome of sperm (fresh and cryopreserved), processed in the absence and presence of 0.5 mg/ml CLC in the addra gazelle. The proteome of Bos taurus, the closest domestic relative, was used as a reference. Mass spectrometry analysis of the addra gazelle sperm proteome revealed 287 proteins. The concentrations of 85 proteins differed between fresh and frozen/thawed samples; nearly all were decreased. Most were associated with metabolic processes, specifically glycolysis, which may explain the decrease in post-thaw motility observed in this species. CLC pretreatment partially prevented the loss of various proteins involved in metabolism including CAPZB (gene = CAPZB), HS90A (gene = HSP90AA1), and PGAM2 (gene = PGAM2). To our knowledge, this is the first study to evaluate the proteome of any wild bovids' sperm, and the first to compare protein levels in sperm pretreated with CLC.

Non-invasive hormonal characterization of the ovarian cycle, pregnancy, and seasonal anestrus of the female addra gazelle (Nanger dama ruficollis).

Non-invasive fecal hormone metabolite monitoring was used to characterize the estrous cycle, pregnancy, and seasonal anestrus of the critically endangered addra gazelle (Nanger dama ruficollis). With less than 250 animals remaining in the wild and ∼168 individuals managed in captivity, it is crucial to maintain sustainable populations. Progestogen and estrogen profiles were obtained from analysis of fecal samples collected approximately every other day, within varying intervals, over the course of 7 years (n = 8 adult females). Average estrous cycle length was 19.5 ± 0.4 days (range, 14-26 days), with a luteal phase length of 14.6 ± 1.2 days (range, 10-16 days), and an inter-luteal period of 5.2 ± 1.4 days (range, 2-7 days). Mean gestation length for six pregnancies was 200.7 ± 0.4 days (range, 200-202 days). Fecal progestogens increased at 12 weeks of gestation and remained elevated until parturition. Addra gazelle females exhibited a period of seasonal anestrus with consistently low progestogen concentrations and no cyclic activity from about September to March. Analysis of reproductive and climate records demonstrated a peak in U.S. births that coincided with maximal rainfall in the native habitat of the addra gazelle. Results show that estrous cycle, luteal phase, and inter-luteal phase lengths in addra are similar to those observed in other gazelle species, however, to our knowledge, this is the first study to demonstrate seasonal anestrus in the Nanger genus.

Pretreatment of Addra gazelle (Nanger dama ruficollis) spermatozoa with cholesterol-loaded cyclodextrins improves cryosurvival.

Preserving genetic diversity of the critically endangered Addra gazelle (Nanger dama ruficollis) could be enhanced through the use of frozen-thawed sperm and artificial insemination. Our aim was to characterize Addra ejaculate traits and to assess the effects of cholesterol-loaded cyclodextrin (CLC) on sperm cryosurvival. Fresh ejaculates were treated with CLC (0, 0.5, 1.5, 3.0, and 6.0 mg/ml) prior to cryopreservation. All males produced spermic ejaculates with >75% sperm motility. The mean ± SEM seminal volume, sperm concentration, percent motility, forward progression, and percent morphologically normal spermatozoa were 3.2 ± 0.3 ml, 1.2 ± 0.3 × 109, 75.82 ± 2.7%, 3.2 ± 0.3 (0-5 scale; 5 = most progressive), and 57.12 ± 3.8%, respectively. More than 92% contained an intact acrosome. There was no effect of time or in vitro incubation on progression or acrosomal integrity on thawed samples (P > 0.05). Spermatozoa pre-treated with 0.5 mg/ml CLC retained higher (P < 0.05) motility post-thaw than aliquots treated with 0, 3.0, or 6.0 mg/ml of CLC. Spermatozoa pre-treated with 0.5, 1.5, or 3.0 mg/ml CLC exhibited greater viability than counterparts (P < 0.05). Sperm kinetics including beat cross frequency (BCF), average path velocity (VAP), curvilinear velocity (VCL), and straight line velocity (VSL) did not differ among samples (P > 0.05). Linearity (LIN) and straightness (STR) were different among samples after thawing. Results demonstrate treatment with CLC (0.5 mg/ml) protects Addra spermatozoa from cryo-damage. Reported advances will facilitate establishment of a frozen repository and support the genetic management of this critically endangered north African desert antelope.

Expression and regulation of Kit ligand in the ovary of the hen.

The Kit system, composed of Kit ligand (KL) and its tyrosine kinase receptor, cKit, has been well characterized in mammals. Studies have shown that it is involved in signaling between the oocyte and somatic cells during the process of follicle maturation. We characterized KL mRNA expression during follicle maturation in the domestic hen, examined regulation of KL and a possible function of the Kit system. KL mRNA expression was assessed using quantitative PCR (n=4 replicates) in follicles of various sizes (1, 3, 5, 6-12 mm, F1). Expression of KL mRNA decreased significantly (p<0.01) with follicle development and was highest in <1 mm follicles, which contained the theca as well as granulosa layers, with high levels also found in the granulosa layer of 3 mm follicles and ovarian stroma. To study regulation of KL mRNA, granulosa cells from 6-8 mm follicles (n=4 replicates) were plated in M199 plus 0.1% BSA in the presence of various treatments including: oocyte conditioned medium (OCM), Vitamin D(3), FSH, estradiol, progesterone and testosterone. OCM caused a dose-related increase (p<0.05) in expression of KL mRNA; Vitamin D(3) increased and FSH decreased expression of KL mRNA. cKit was detected (at the expected size) in the theca layer of 3-5 mm follicles and in a lysate of whole <1mm follicles. Culture of granulosa cells in the presence of OCM resulted in a decrease of P4 secretion, an effect blocked by pre-incubation of OCM with cKit antibody. Although OCM caused a dose-related increase in E2 secretion from theca, this was not blocked by cKit antibody.

Vitamin D regulates anti-Mullerian hormone expression in granulosa cells of the hen.

Anti-Mullerian hormone (AMH) is involved in the regression of the Mullerian ducts in mammalian and avian male embryos as well as the right oviduct in avian female embryos. AMH is expressed by granulosa cells of adult hens and mammals and is thought to be involved in the recruitment of follicles from the primordial pool as well as in regulating follicle-stimulating hormone (FSH) sensitivity. We have shown that AMH expression by the granulosa layer of hens is high in the small follicles but decreased in the larger hierarchical follicles. The decline in expression of AMH with increasing follicle size is associated with an increase in expression of the receptor for FSH (FSHR) in the granulosa layer, although the mechanism is not known. In this study, we tested whether vitamin D (1,25-dihydroxyvitamin D3) regulates expression of AMH mRNA in granulosa cells of the hen. Granulosa cell layers were removed from follicles 3-5 mm and 6-8 mm in size, dispersed, and cultured for 24 h in Medium 199 + 5% fetal bovine serum (n = 7). The medium was removed and replaced with Medium 199 + 0.1% bovine serum albumin and vitamin D (at doses of 0, 10, and 100 nM) and cultured for 24 h. Cells were harvested and RNA was extracted for use in quantitative PCR. Parallel 96-well plates were set up to examine cell proliferation. AMH and FSHR mRNA expressions were evaluated, and all values were standardized to 18S reactions. There was a significant (P < 0.05) dose-related decrease in the expression of AMH mRNA in granulosa cells of 3- to 5-mm and 6- to 8-mm follicles in response to vitamin D. Additionally, FSHR mRNA and cell proliferation were significantly (P < 0.05) increased by vitamin D in both groups. Western blot analysis for the vitamin D receptor (VDR) showed doublet bands at the expected sizes (58 and 60 kDa) in protein isolated from the chicken granulosa layer. Immunohistochemistry was used to identify VDR within the follicle, and it predominantly localized to the nucleus of granulosa cells. VDR mRNA expression in the granulosa layer, relative to follicle development, was increased (n = 4; P < 0.05) with follicle development, with greatest expression in the F1 follicle. There was no evidence for expression (mRNA or protein) of the calcium-binding protein, calbindin (CALB1), in the ovary or granulosa layer. Overall, these results suggest that vitamin D regulates AMH expression, and thereby may influence follicle selection in the hen.