Irregular ovarian cyclicity is associated with adrenal activity in female eastern black rhinoceros (Diceros bicornis michaeli).

To achieve self-sustaining and genetically diverse populations ex situ, captive breeding programmes must ensure good overall rates of reproduction, and equal contribution across individuals. Previous research in the critically endangered eastern black rhinoceros (Diceros bicornis michaeli) revealed a high incidence of irregular oestrous cyclicity; in particular extended cycle duration among nulliparous females and acyclic periods in parous females that have not bred for several years. Irregular ovarian activity could play a role in reduced reproductive output; however, the mechanisms underlying these anomalies are poorly understood. The aim of this study was to measure faecal glucocorticoid metabolite (fGCM) concentrations and variability prior to and during periods of regular and irregular ovarian activity, and determine if adrenal activity influences the occurrence of different cycle types in this species. Faecal samples were collected every other day from parous (N = 6) and nulliparous (N = 12) females at eight European institutions for periods of 9-15 months. Concentration and variability in fGCM were compared between periods of regular and irregular cyclicity and between different cycle types (<20 days, 20-40 days, >40 days, acyclic) using generalized linear mixed models. Concentrations of fGCM were influenced by season and higher during the luteal than the follicular phase of the oestrous cycle. Taking this into account, fGCMs were increased during periods of irregular cyclicity (all types combined and during cycles >40 days in length) compared to 20-40 day cycles. This was predominantly driven by nulliparous females. The variation in fGCM concentration also differed between periods of regular and irregular cyclicity; higher standard deviation in fGCM preceded irregular cycles and >40 day cycles compared to 20-40 day cycles. These results suggest that although fGCM concentrations fluctuate across the oestrous cycle in this species, changes in adrenal activity at specific times could be one factor associated with irregular ovarian activity in the black rhinoceros.

Male reproductive success is correlated with testosterone in the eastern black rhinoceros (Diceros bicornis michaeli).

Among natural populations of polygynous species, males often vary in their lifetime reproductive success. However, in managed populations of endangered species, either in situ or as part of captive breeding programmes, it is important to understand why differences in reproductive success occur. The European captive population of the critically endangered eastern black rhinoceros is currently under-performing relative to their wild counterparts, with low reproductive output and high reproductive skew limiting growth and genetic diversity. To investigate why over 40% of captive males fail to breed, faecal samples were collected weekly from 23 males at 12 institutions across Europe for 4-32 months. Testosterone metabolite concentration was compared between proven and non-proven males and a number of intrinsic and extrinsic factors that could influence reproductive success were also investigated. Males that sired within the last 3½ years had significantly higher androgen concentrations than non-proven males, and average testosterone was positively correlated with the number of offspring sired per year spent in the reproductive age class. Proven and non-proven males did not differ in their body condition, or in average faecal glucocorticoid concentration. Differences in individual temperament were associated with adrenal activity, but did not correlate with reproductive category. Highest testosterone concentrations were observed in proven males that were housed with females during oestrus, and lowest concentrations in non-proven females not housed with females at all during the study period. Further work is necessary to determine whether proven males had higher testosterone due to underlying differences associated with quality, or whether external stimuli such as access to females could influence testosterone concentration and increase a male's chances of becoming a successful breeder.

Irregular ovarian activity, body condition and behavioural differences are associated with reproductive success in female eastern black rhinoceros (Diceros bicornis michaeli).

Ex situ populations of endangered species such as the black rhinoceros play an important role in global conservation strategies. However, the European captive population of eastern black rhinoceros is performing sub-optimally, with growth rates and genetic viability limited by low birth rates and high reproductive skew. We investigated several intrinsic differences between parous and nulliparous females that may underlie differences in reproductive success, including ovarian cyclicity, adrenal activity, behaviour and body condition. Faecal samples were collected from 39 females (17 parous, 15 nulliparous and 7 pre-reproductive) at 11 zoological institutions, every other day for between 4months and 6years. Progestagen metabolite concentration indicated that although all non-pregnant females exhibited ovarian activity, irregular cyclicity was common. Longer cycles (>40days) were more common in nulliparous females and periods of acyclicity observed more often in females that had not bred for at least 7years. Even when endocrine data indicated clear ovarian activity, overt behavioural signs of oestrus were not always apparent, particularly among nulliparous females. Faecal glucocorticoids did not differ between parous and nulliparous females, although did differ according to individual temperament. More unpredictable temperaments were associated with higher glucocorticoids, and nulliparous females tended to be rated as more unpredictable. Finally, nulliparous females had higher body condition scores than parous females. This is the first comprehensive survey of the reproductive physiology of this European captive population, and highlights a number of intrinsic differences related to parity, which may underlie differences in reproductive success among captive female black rhinoceros.

Salivary glands act as mucosal inductive sites via the formation of ectopic germinal centers after site-restricted MCMV infection.

We investigated the hypothesis that salivary gland inoculation stimulates formation of ectopic germinal centers (GCs), transforming the gland into a mucosal inductive site. Intraglandular infection of mice with murine cytomegalovirus (MCMV; control: UV-inactivated MCMV) induces salivary gland ectopic follicles comprising cognate interactions between CD4(+) and B220(+) lymphocytes, IgM(+) and isotype-switched IgG(+) and IgA(+) B cells, antigen presenting cells, and follicular dendritic cells. B cells coexpressed the GC markers GCT (57%) and GL7 (52%), and bound the lectin peanut agglutinin. Lymphoid follicles were characterized by a 2- to 3-fold increase in mRNA for CXCL13 (lymphoid neogenesis), syndecan-1 (plasma cells), Blimp-1 (plasma cell development/differentiation), and a 2- to 6-fold increase for activation-induced cytidine deaminase, PAX5, and the nonexcised rearranged DNA of an IgA class-switch event, supporting somatic hypermutation and class-switch recombination within the salivary follicles. Intraglandular inoculation also provided protection against a systemic MCMV challenge, as evidenced by decreased viral titers (10(5) plaque-forming units to undetectable), and restoration of normal salivary flow rates from a 6-fold decrease. Therefore, these features suggest that the salivary gland participates in oral mucosal immunity via generation of ectopic GCs, which function as ectopic mucosal inductive sites.

A focused salivary gland infection with attenuated MCMV: an animal model with prevention of pathology associated with systemic MCMV infection.

While the salivary gland has been recognized as an important effector site of the common mucosal immune system, a useful model for studying anti-viral salivary gland immune responses in vivo and for exploring the role of the salivary gland within the common mucosal system has been lacking. Murine cytomegalovirus (MCMV) is a beta-herpesvirus that displays a strong tropism for the salivary gland and produces significant morbidity in susceptible mice when introduced by intraperitoneal (i.p.) inoculation. This study tested the hypothesis that MCMV morbidity and pathology could be reduced by injecting the virus directly the submandibular salivary gland (intraglandular (i.g.)), using either in vivo derived MCMV or the less virulent, tissue-culture-derived MCMV (tcMCMV). Peak salivary gland viral titers were completely unaffected by infection route (i.p vs. i.g.) after inoculation with either MCMV or tcMCMV. However, i.g. tcMCMV inoculation reduced viremia in all systemic tissues tested compared to i.p. inoculation. Furthermore, systemic organ pathology observed in the liver and spleen after i.p. inoculation with either MCMV or tcMCMV was completely eliminated by i.g. inoculation with tcMCMV. Cellular infiltrates in the salivary glands, after i.p. or i.g. inoculation were composed of both B and T cells, indicating the potential for a local immune response to occur in the salivary gland. These results demonstrate that a focused MCMV infection of the salivary gland without systemic organ pathology is possible using i.g. delivery of tcMCMV.

Reovirus serotype 1/strain Lang-stimulated activation of antigen-specific T lymphocytes in Peyer's patches and distal gut-mucosal sites: activation status and cytotoxic mechanisms.

Intraduodenal priming of mice with reovirus serotype 1/strain Lang (reovirus 1/L) stimulates gut lymphocytes and generates precursor and effector CTLs. Our earlier studies demonstrated that germinal center and T cell Ag (GCT) is a marker which identifies reovirus 1/L-specific precursor CTL and effector CTL in Peyer's patches (PP) of reovirus 1/L-inoculated mice. In this study, we characterized the expression of the activation markers, GCT and CD11c, on reovirus 1/L-stimulated gut lymphocytes and the effector mechanisms involved in reovirus 1/L-specific cytotoxicity. We found that intraduodenal reovirus 1/L inoculation of mice induced the expression of both GCT and CD11c on PP lymphocytes (PPL), intraepithelial lymphocytes (IEL), and lamina propria lymphocytes (LPL), and these activated cells expressed Fas ligand (FasL). The majority of the GCT+ CD11c+ IEL and LPL expressed a phenotype, TCRalphabeta+ Thy-1+ CD8+ similar to that expressed on reovirus 1/L-stimulated PPL. However, splenic lymphocytes expressed GCT but not CD11c after stimulation with reovirus 1/L. Perforin, Fas-FasL, and TRAIL pathways were found to be involved in PPL, IEL, and LPL cytotoxic activity against reovirus 1/L-infected targets. In PPL, perforin and Fas-FasL pathways were more effective than TRAIL. In IEL, all three cytotoxic mechanisms were equally as effective. However, LPL prefer Fas-FasL and TRAIL over perforin. Further, we demonstrated the preferential migration of GCT+ PPL to the intraepithelial compartment and the lamina propria. These results suggest that GCT and CD11c can be used as activation markers for gut lymphocytes and CD11c can also be used to differentiate between activated gut and systemic lymphocytes.