Non-invasive identification of protein biomarkers for early pregnancy diagnosis in the cheetah (<i>Acinonyx jubatus</i>)

<div><p>Approximately 80% of cheetahs living in typical zoological collections never reproduce. In more than 60% of breedings, the female is confirmed to ovulate, but parturition fails to occur. It is unknown if these non-pregnant intervals of elevated progesterone (deemed luteal phases) are conception failures or a pregnancy terminating in embryonic/fetal loss. There have been recent advances in metabolic profiling and proteome analyses in many species with mass spectrometry used to identify ‘biomarkers’ and mechanisms indicative of specific physiological states (including pregnancy). Here, we hypothesized that protein expression in voided cheetah feces varied depending on pregnancy status. We: 1) identified the expansive protein profile present in fecal material of females; and 2) isolated proteins that may be candidates playing a role in early pregnancy establishment and diagnosis. Five hundred and seventy unique proteins were discovered among samples from pregnant (n = 8), non-pregnant, luteal phase (n = 5), and non-ovulatory control (n = 5) cheetahs. Four protein candidates were isolated that were significantly up-regulated and two were down-regulated in samples from pregnant compared to non-pregnant or control counterparts. One up-regulated candidate, immunoglobulin J chain (IGJ; an important component of the secretory immune system) was detected using a commercially available antibody via immunoblotting. Findings revealed that increased IGJ abundance could be used to detect pregnancy successfully in >80% of 23 assessed females within 4 weeks after mating. The discovery of a novel fecal pregnancy marker improves the ability to determine reproductive, especially gestational, status in cheetahs managed in an <i>ex situ</i> insurance and source population.</p></div

Non-invasive identification of protein biomarkers for early pregnancy diagnosis in the cheetah (<i>Acinonyx jubatus</i>) - Fig 4

<p><b>Western blot intensities for protein candidates: a) immunoglobulin J chain; b) trefoil factor 3; c) complement C3 (iC3b fragment); d) complement C3 (C3dg fragment); e) alkaline phosphatase; and f) myosin binding protein C.</b> Source was fecal extracts from female cheetahs during the first 4 wk of pregnancy (n = 8), a non-pregnant luteal phase (n = 5), or non-ovulatory controls (n = 5). Red dots indicate outliers (i.e., >1.5 interquartile ranges above the third quartile or below the first quartile). Asterisk indicates difference trend (<i>P</i> < 0.1).</p

Protein identification and relative quantification ratios (± SEM) of pregnancy biomarker candidates (n = 6) from cheetah fecal extracts in two mass spectrometry analyses.

<p>Protein identification and relative quantification ratios (± SEM) of pregnancy biomarker candidates (n = 6) from cheetah fecal extracts in two mass spectrometry analyses.</p

Specific information for antibodies used in western blotting of fecal proteins from pregnant and non-pregnant cheetahs.

<p>Specific information for antibodies used in western blotting of fecal proteins from pregnant and non-pregnant cheetahs.</p

Representative fecal progestogen metabolite profiles of four cheetahs that were pregnant or experiencing a non-pregnant luteal phase (after gonadotropin treatment with LH/hCG to stimulate ovulation).

<p>Arrow indicates approximate termination of elevated progestogens associated with a non-pregnant luteal phase.</p

Intensity of immunoglobulin J chain expression for cheetahs that were pregnant (n = 14) or in a non-pregnant luteal phase (n = 9) compared to samples from the same females during a non-ovulatory control period.

<p>Each bar represents a single female. Inset representative blot image of samples from the same four pregnant females during a non-ovulatory period. Asterisks indicate instances when immunoglobulin J chain expression did not change between ovulatory and non-ovulatory states to allow accurate pregnancy determination.</p

Mean (± SEM) total protein and steroid hormone metabolite concentrations of fecal extracts from female cheetahs.

<p>Protein was extracted from pooled samples collected over 28 d and steroid hormone metabolites extracted from individual fecal samples collected over 8 to 13 wk. Bars with different letters within a metric were different (<i>P</i> < 0.05).</p

Workflow used to reduce total protein output lists from cheetah fecal samples analyzed with mass spectrometry in two sets: 1) pregnant (n = 5) paired with non-ovulatory (control) samples (n = 5) from the same females; and 2) pregnant (n = 5) paired with matched samples from other non-pregnant females experiencing a luteal phase (n = 5).

<p>Samples were labeled with reporter ions for relative quantification of each protein between pregnant and non-pregnant reproductive groups. From all ‘identified’ proteins in both lists, those proteins failing to produce at least two unique peptides for relative quantitation were deleted from further consideration, leaving only those that could be ‘quantified’. After comparing individual protein quantifications between the five pregnant and non-pregnant paired samples in each analysis, lists were further reduced to include proteins with expression differences that were significant. Finally, only those proteins were retained that were consistent in over- or under-expression in all pregnant versus non-pregnant sample comparisons. Black arrows indicate increased or decreased protein expression in pregnant versus non-pregnant (i.e., non-ovulatory or non-pregnant luteal) samples.</p

Mean (± SEM) seasonal ejaculate traits from cheetahs where semen was collected in different seasons at institutions throughout the USA (169 total ejaculates; number/season indicated in parentheses).

<p>There were no differences (<i>P</i> > 0.05) in any trait across seasons.</p

Motile Sperm Output by Male Cheetahs (<i>Acinonyx jubatus</i>) Managed <i>Ex Situ</i> Is Influenced by Public Exposure and Number of Care-Givers

<div><p>The collective cheetah (<i>Acinonyx jubatus</i>) population in zoological institutions has never been self-sustaining because of challenges in natural reproduction. A retrospective analysis of North American zoo-breeding records has revealed that >90% of litters produced since 2003 occurred in facilities ‘off-display’ from the public. We examined seminal, endocrine, and behavioral traits of 29 adult male cheetahs that were: 1) managed in public exhibit or off-display facilities; 2) maintained by different numbers of cheetah-specific care-givers; and 3) living adjacent to varying numbers of adult conspecifics. Cheetahs housed off-display produced more total motile sperm/ejaculate (<i>P</i> = 0.04) than on-exhibit males. This finding was mirrored in our laboratory’s historical records where two-fold more total motile sperm (<i>P</i> < 0.01) were measured in ejaculates from individuals with no public exposure (n = 43) compared to on-exhibit (n = 116) counterparts. Males at institutions with ≤3 care-givers also produced more total motile sperm/ejaculate (<i>P</i> < 0.03) and spent more time behaviorally active (<i>P</i> < 0.01) than at facilities using >3 care-givers. Exposure to high numbers of conspecifics within the same institution did not impact (<i>P</i> > 0.05) seminal traits, and presence of the public, care-giver number, or animals/facility had no influence (<i>P</i> > 0.05) on androgen or glucocorticoid excretion or other behavioral metrics. Findings indicate that male cheetahs are sensitive to general public exposure and too many care-givers, resulting in compromised motile sperm output/ejaculate with mechanism of action unrelated to altered androgen or glucocorticoid excretion.</p></div