Bronchoalveolar lavage for diagnosis of tuberculosis infection in elephants.

Tuberculosis (TB) has been known to affect elephants for thousands of years. It was put into spotlight when few circus elephants were diagnosed carrying Mycobacterium (M.) tuberculosis. Because of the zoonotic risk and high susceptibility to M. tuberculosis, periodic testing was enacted since, in captive breeding programmes. Presently, trunk wash is the recommended diagnostic procedure for TB. Trunk wash, however, puts the operator at risk, has low sensitivity, and is prone to contamination. Here, bronchoalveolar lavage is described for the first time for TB diagnosis in elephants. Bronchial, trunk and mouth fluids were investigated using bacterial culture, M. tuberculosis complex (MTC)-specific real-time quantitative PCR (qPCR) and mycobacterial genus-specific qPCR for overall presence of mycobacteria or mycobacterial DNA including bacteria or DNA of closely related genera, respectively, in 14 elephants. Neither bacteria of the MTC nor their DNA were identified in any of the elephants. Yet, 25% of the cultures grew non-tuberculous mycobacteria (NTM) or closely related bacterial species. Furthermore, 85% of the samples contained DNA of NTM or closely related bacterial genera. This finding might explain continued false-positive results from various serological tests. From a zoonotic point of view, bronchoalveolar lavage is safer for the testing personal, has higher probability of capturing MTC and, through PCR, identifies DNA NTM in elephants. Yet, necessary endoscopic equipment, animal sedation and access to a TB reference laboratory might pose challenging requirements in remote conditions in some elephant range countries.

Life cycle of feline Corpora lutea: histological and intraluteal hormone analysis.

The corpus luteum (CL) is a transient hormone gland on the ovary that produces progesterone (P4) for the maintenance of pregnancy. It develops from residual follicular granulosa and theca cells after ovulation. Very little is known about the cellular and hormonal processes within CLs obtained from pregnant and pseudopregnant felids. Therefore, our aim was to review the luteal function in feline CLs of different reproductive stages in conjunction with our data obtained in domestic cats and Eurasian lynxes. Corpus luteum function in lynxes is of particular interest, as a post-partum luteal activity was suggested based on repeated ultrasonography and endocrine examinations. Histology of CL from pregnant and pseudopregnant domestic cats clearly reflects the luteal function. The formation of the CL after ovulation is characterized by transforming of theca and granulosa cells into steroidogenic luteal cells and is accompanied by increased intraluteal and circulating P4 levels. Luteal regression is steadily progressive; the first signs (coarsed vacuolization, increased proportion of non-steroidogenic cells) are visible already in CL from the second trimester of pregnancy.

MALDI-TOF "fingerprint" phospholipid mass spectra allow the differentiation between ruminantia and feloideae spermatozoa.

The detailed comparative analysis of sperm lipids could essentially contribute to a better understanding of membrane function in the context of fertilization and, moreover, of sperm preservation. The application of sensitive analytical methods is particularly necessary for endangered species as the available amount of spermatozoa (and, accordingly, extractable lipids) is strongly limited. It will be shown that matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) is a fast, simple and sensitive method for the determination of the phospholipid composition of spermatozoa from several ruminantia (cattle, roe deer, Klipspringer) and feloideae species (domestic cat, Siberian tiger, fosa). Characteristic "fingerprints" are obtained from the positive ion spectra that allow the differentiation between both animal groups. In contrast to the lipid extracts of ruminantia spermatozoa which predominantly contain ether lipids including essential amounts of plasmalogens, the more complex phospholipid composition of feloideae spermatozoa is clearly dominated by diacyl phospholipids and contains only marginal amounts of plasmalogens. It will also be shown that the lipid compositions of ejaculated, electroejaculated and cauda epididymal spermatozoa of the same species are very similar and give comparable data. Therefore, the analysis of ejaculated spermatozoa is not an absolute must.

First successful artificial insemination with frozen-thawed semen in rhinoceros.

The first successful artificial insemination (AI) in a rhinoceros was reported in 2007 using fresh semen. Following that success, we decided to evaluate the possibility of using frozen-thawed semen for artificial insemination. Semen, collected from a 35-36 year old Southern white rhinoceros (Ceratotherium simum simum) in the UK was frozen using the directional freezing technique. This frozen semen was used in two intrauterine AI attempts on a 30 years old female rhinoceros in Hungary. The first attempt, conducted 30 days postpartum with an insemination dose of approximately 135 x 10(6) motile cells, failed. The second attempt, conducted two estrus cycles later with an insemination dose of approximately 500 x 10(6) motile cells, resulted in pregnancy and the birth of a healthy offspring. This represents the first successful AI using frozen-thawed semen in a rhinoceros, putting it among very few wildlife species in which AI with frozen-thawed semen resulted in a live birth. The incorporation of AI with frozen-thawed semen into the assisted reproduction toolbox opens the way to preserve and transport semen between distant individuals in captivity or between wild and captive populations, without the need to transport stressed or potentially disease carrying animals. In addition, cryopreserved spermatozoa, in combination with AI, are useful methods to extend the reproductive lifespan of individuals beyond their biological lifespan and an important tool for managing genetic diversity in these endangered mammals.

Pregnancy diagnosis in urine of Iberian lynx (Lynx pardinus).

Diagnosis of pregnancies is an important management tool for the Iberian lynx Conservation Breeding Program, a program geared to recover the world's most endangered felid. Non-invasive methods such as fecal hormone analyses are not applicable to the lynx, since fecal progestin does not follow the typical pregnancy pattern of felids. Therefore, we aimed to test whether urine can be used as an alternative substance for pregnancy diagnosis in the Iberian lynx. Progesterone immunoreactive metabolites were determined in urine samples of pregnant and non-pregnant females before and during breeding season. Additionally, we used the Witness Relaxin test to determine relaxin in blood and urine. No differences were found in progestin concentrations determined in urine samples collected from pregnant and non-pregnant animals between day 1 and 65 following mating. Although the Witness Relaxin test was positive in serum samples collected from animals between day 32 and 56 of pregnancy, it failed in both fresh and frozen urine samples collected from the same stage of pregnancy. A weak relaxin reaction in urine samples collected from animals between day 29 and 46 of pregnancy was detectable after urines were concentrated by ultrafiltration (>50x). Concentrated samples obtained from non-pregnant and early pregnant animals yielded negative test results. In conclusion, the Witness Relaxin test can be applied for pregnancy diagnosis in Iberian lynx in both serum and concentrated urine samples obtained during the second half of pregnancy. A positive relaxin test indicates an ongoing pregnancy, whereas negative tests must be judged carefully as hormone concentrations might be below detection thresholds.

Seasonal profiles of ovarian activity in Iberian lynx (Lynx pardinus) based on urinary hormone metabolite analyses.

The Iberian Lynx Ex-Situ Conservation Programme is an essential part of a co-ordinated action plan to conserve the most endangered felid species of the world. Successful captive breeding demands reliable methods for reproduction monitoring including reliable non-invasive pregnancy diagnosis. During a 3-year study, urine samples from six captive Iberian lynx females were obtained (one non-pregnant, one pseudo-pregnant and 11 pregnant cycles). Progesterone, pregnanediol and oestradiol were determined in urinary extracts and relevant urinary oestrogen metabolites were characterized by high-performance liquid chromatography (HPLC). Urinary progestins did not follow the typical pregnancy-related course of felids. In the lynx, we failed to demonstrate an urinary progestin elevation during pregnancy. In contrast, urinary oestrogens increased from 3.8 +/- 0.6 to 8.6 +/- 0.5 ng/mg creatinine (p < 0.001) during the pregnancy. A comparison of pseudo-pregnant with pregnant cycles revealed a further increase of oestrogens caused by implantation (p < 0.05). In one female, which refused to mate, no difference was estimated between oestrogens levels during the breeding and non-breeding seasons. Almost 10-fold higher oestrogen concentrations were measured in urines of females that shared enclosures with males. HPLC analysis of oestrogens in urine samples collected from Iberian lynx during the pregnancy revealed that lynx urine is composed of two polar oestrogen metabolites in addition to oestrone and minor amounts of oestradiol. Oestrone was detectable in all urinary extracts (8-12% of metabolites), whereas oestradiol was elevated only during late pregnancy (18%). Thus, seasonal luteal activity in Iberian lynx can be monitored by urinary oestrogens. The increase of urinary oestradiol during late pregnancy might indicate an oestradiol secretion by the lynx placenta.

Pregnancy monitoring in dogs and cats using 3D and 4D ultrasonography.

Three-dimensional (3D)/four-dimensional (4D) volume ultrasound is an established method in human medicine that offers various options for analysing and presenting ultrasound volume data. However, the successful application of the different 3D/4D imaging modalities in pregnant dogs and cats has not yet been reported in the literature. The main reasons for this are: (1) the high costs of 3D/4D ultrasound systems, (2) operation difficulties due to high breathing frequency in non-sedated animals and (3) the missing specific knowledge in veterinary medicine concerning how to perform high-quality volume scans. Automatically acquired ultrasound volume data sets were generated with two different ultrasound systems: the portable Voluson i and the stationary Voluson Expert 730. Different 3D/4D imaging modalities were tested in regard of their practicability in pregnancy monitoring in dogs and cats. Nine different volume imaging modalities were applied using the saved files. For the presentation of the static 3D volume data sets, we used the multiplanar, niche, surface, transparency, glass body, inversion, volume calculation and tomographic ultrasound imaging modes. For the dynamic 4D data, the surface and glass body modes were applied. By changing the human standard settings to the requirements of small animal anatomy, it was found that 3D/4D ultrasound has great potential for the characterization of pregnancy in queens and bitches. The 3D/4D technology offered advanced information about pregnancy status and birth prediction and improved the diagnostic confidence. By using standardized examination protocols, 3D/4D ultrasound will allow a reduction in examination time by generating even more relevant information. These benefits, combined with possible future cost reduction of commercial ultrasound systems, might lead to frequent utilization in routine pregnancy diagnostic and birth management in small animal practice.

Non cat-like ovarian cycle in the Eurasian and the Iberian lynx - ultrasonographical and endocrinological analysis.

The Iberian lynx is considered the most endangered felid species. Therefore, an ex situ conservation program was initiated to protect this species from extinction. Additional knowledge on lynx reproduction biology and reliable methods for reproductive monitoring are important for developing a captive breeding program. The aim of this study in lynx was to implement transrectal ultrasonography to visualize ovarian structures (follicles, corpora lutea) and to assess ovarian activity in addition to analysis of serum progesterone and oestradiol. Because of limited access to Iberian lynxes, the less-endangered Eurasian lynx and bobcat were also studied in this comparative study. Recent endocrinological studies based on faecal and urinary progesterone and oestrogen metabolites revealed that steroid profiles in both these species were alike and did not follow the typical pattern of other felids. Pregnancy diagnosis was not possible, since progesterone concentrations did not differ between pregnant and pseudopregnant animals. Progesterone was also detected after parturition as well as after weaning until the onset of a new oestrous cycle. In the present study, the presence of corpora lutea during the non-breeding season was confirmed by ultrasonography and by elevated serum levels of progesterone averaging 3.56 +/- 1.3 ng/ml in Eurasian and 6.1 +/- 0.26 ng/ml in Iberian lynx, respectively. The ultrasonographical findings on the ovarian structures suggest strongly that corpora lutea developed after ovulation stay active until November and regress before the onset of the next oestrus.

Early embryo development in the elephant assessed by serial ultrasound examinations.

The elephant has an extraordinary long pregnancy, lasting 21 months. However, knowledge on embryo development is limited. To date, only single morphological observations of elephant embryo development associated with placentation are available, all lacking correlation to gestational age. The present study describes morphological characteristics of early embryo development in the elephant with exact biometric staging. Six pregnancies in five Asian and one African elephants with known conception dates were followed by 2D and 3D ultrasound, covering the embryonic period from ovulation to day 116 post-ovulation. The embryonic vesicle was earliest observed was on day 50 p.o. The proper embryo was not detected until day 62 p.o. Embryonic heartbeat was first observed on day 71 p.o. The allantois, which became visible as a single sacculation on day 71 p.o. was subdivided in four compartments on day 76 p.o. By day 95 p.o., head, rump, front and hind legs were clearly distinguished. Between days 95 and 103 p.o. the choriovitelline placenta was replaced by the chorioallantoic placenta. A physiological midgut herniation was transiently present between days 95 and 116 p.o. On the basis of the late appearance of the embryonic vesicle, delayed implantation in the elephant is discussed. The study provides a coherent description of elephant embryonic development, formation of the extraembryonic organs and their role in placenta formation, all of which are of interest for both comparative evolutionary studies and the improvement of assisted reproduction techniques.

Obstetrics in elephants.

Obstetrics, one of the oldest fields in veterinary medicine, is well described and practiced in domestic and exotic animals. However, when providing care during elephant birth or dystocia, veterinary intervention options differ greatly from any domestic species, and are far more limited due to the dimensions and specific anatomy of the elephant reproductive tract. In addition, aging of captive elephant populations and advanced age of primiparous females make active birth management increasingly important. Intrauterine infection, uterine inertia and urogenital tract pathologies are emerging as major causes for dystocia, often leading to foetal and dam death. This paper reviews the current knowledge on elephant birth and the factors associated with dystocia. It then summarises recommendations for birth and dystocia management. As Caesarean section, the most common ultima ratio in domestic animal obstetrics, is lethal and therefore not an option in the elephant, non-invasive medical treatment, induction of the Fergusson reflex or the conscious decision to leave a retained foetus until it is expelled voluntarily, are key elements in elephant obstetrics. Surgical strategies such as episiotomy and foetotomy are sometimes inevitable in order to try to save the life of the dam, however, these interventions result in chronic post-surgical complications or even fatal outcome. Limited reliable data on serum calcium concentrations, and pharmacokinetics and effect of exogenous oestrogen, oxytocin, and prostaglandins during birth provide the scope of future research, necessary to advance scientific knowledge on obstetrics in elephants.

Non-invasive monitoring of hormones: a tool to improve reproduction in captive breeding of the Eurasian lynx.

The survival of many critical endangered mammal species is often depending on successful captive breeding programmes which include the future option of reintroduction to the wild. Breeding in captivity also demands the application of modern assisted reproductive techniques to ensure maximal biodiversity, but knowledge on reproductive physiology is often limited. Therefore, non-invasive monitoring of urinary and faecal hormones has become an important tool for reproductive management. To exemplify the importance of non-invasive hormone monitoring, we choose the Eurasian lynx as a model for the world's most endangered felid species, the Iberian lynx. We analysed faecal samples of pregnant and pseudo-pregnant female Eurasian lynxes during a 3-year study period. Compared to pre-mating levels faecal progesterone metabolite profiles revealed a tendency towards higher levels in pregnant and pseudo-pregnant females with no difference between both categories. Oestrogen levels raised in both pregnant and pseudo-pregnant females with a tendency to be more elevated and prolonged in pregnant females. Surprisingly both E2 and P4 metabolites were highly correlated (r(2) =0.8131, p < 0.0001) showing a postpartum increase both in pregnant and pseudo-pregnant females. The results from the Eurasian lynx revealed that the measurement of faecal progesterone metabolites led to profiles dissimilar to profiles shown in other felid species, but similar to those from faecal gestagen metabolite analysis in the Iberian lynx. To identify faecal gestagen and oestrogen metabolites a radio-metabolism study was performed. Using the progesterone immunoassay two major progesterone metabolites were detected demonstrating that the assay indeed tracks the relevant metabolites. The oestrogen assay measured authentic 17beta-oestradiol and oestrone, and their conjugates. The analysis of the faecal metabolite composition in samples from early and late pregnancy and lactation particularly revealed a distinct shift in the relation between 17beta-oestradiol and oestrone that changed in favour of oestrone. This might indicate different hormone sources during and after pregnancy (corpus luteum, placenta). We hypothesize, that placental steroid analysis in combination with other highly sophisticated analytical techniques, like liquid chromatography mass spectrometry or urinary relaxin analysis may led to analytical options to confirm pregnancy and to differentiate this from pseudo-pregnancy in lynx species.

Artificial insemination in the anoestrous and the postpartum white rhinoceros using GnRH analogue to induce ovulation.

The objective of this study was to develop AI and to achieve first time pregnancy in a nulliparous rhinoceros. For this, one 24-year-old irregular cycling female white rhinoceros was selected, which had never been mated. The endocrine function was monitored by faecal and serum pregnane analysis. Ultrasound determined the optimal day for AI by measuring follicle sizes of 2.0, 2.6, 3.0, 3.2 cm on days -6, -4, -1, 0 of the induced oestrous cycle, respectively. AI was performed and ovulation induced when a pre-ovulatory-sized follicle was present using GnRH analogue, deslorelin. Fresh semen was deposited in the uterine horn using a patented AI catheter overcoming the hymeneal membrane and torturous cervical folds non-surgically. Moreover, ultrasound monitoring of the uterine involution and ovarian activity on days 16, 26, 30 postpartum facilitated the induction of and the AI on the first postpartum oestrous in a rhinoceros using GnRH analogue. Two consecutive pregnancies were achieved by AI for the first time in the rhinoceros. Pregnancies were diagnosed by elevated serum and faecal 20-oxo-pregnane concentrations. In addition ultrasound measured biometric parameters of the two foetuses on days 86 and 133 of gestation. Two female calves were born after 490 and 502 days of gestation, yet one calf was stillborn. AI in rhinoceros might now be used as assisted reproduction technology tool to boost critically small captive rhinoceros populations.

Assisted reproduction in female rhinoceros and elephants--current status and future perspective.

Over the last few decades, rhinoceroses and elephants became important icons in the saga of wildlife conservation. Recent surveys estimate the wild Asian (Elephas maximus) and African (Loxodonta africana) elephant populations to be, at most, 50 250 and 637 600 respectively. For the five rhinoceros species, black (Diceros bicornis), white (Ceratotherium simum), Indian (Rhinoceros unicornis), Javan (Rhinoceros sondaicus) and Sumatran rhinoceros (Dicerorhinus Sumatrensis), the population estimates of 3610, 11 330, 2400, 60 and 300, respectively, are of even greater concern. Protected against habitat loss, poaching and left undisturbed, rhinoceros and elephants reproduce well in the wild. But small and decreasing populations make successful captive management of these taxa increasingly important. In captivity, however, most populations face possible 'extinction' because of historically poor reproductive performance. From the first descriptions of the reproductive anatomy and the oestrous cycle (Laws 1969; Kassam and Lasley 1981; Balke et al. 1988a,b; Plotka et al. 1988; Godfrey et al. 1991) to the present use of advanced assisted reproduction technologies, researchers have strive to understand the function and dysfunction of the reproductive biology of these charismatic species. This paper reviewed the current knowledge on rhinoceros and elephant reproduction biology, reproductive cycle, gestation, dystocia, reproductive pathology, oestrous induction and artificial insemination, sperm sexing, IVF and contraception, and how this knowledge is or might be used to aid species conservation for maximal reproductive efficiency and enhancement of genetic management.

Comparative endocrine investigations in three bear species based on urinary steroid metabolites and volatiles.

In order to improve breeding of in situ populations of bears, a comprehensive study of reproductive physiology in Brown (Ursus arctos), Spectacled (Tremarctos ornatus) and Giant panda bears (Ailuropoda melanoleuca) was performed. The objective was to perform non-invasive analyses of urinary and fecal steroid metabolites. In addition, we investigated the presence of reproduction-related urinary volatile substances of these bears that might trigger the reproductive behavior. Urinary estrogen concentrations, routinely used to monitor follicular activity in Giant panda, were inappropriate for monitoring follicular activity in Spectacled bear. In addition, no estrogen peak related to mating activity was observed in Brown bear. Further contrasting Giant panda, although urinary pregnanediol analyses failed to indicate luteal activity in either Spectacled or Brown bears, urinary (Spectacled bear) and fecal (Brown bear) concentrations of progesterone were an appropriate indicator of luteal activity. The Giant panda had volatile components (medium-chain fatty acids) in their urine that increased simultaneously with the seasonal increase of estrogens. These fatty acids were also detected in the Brown during estrus and Spectacled bear. Further studies on the behavioral relevance of these fatty acids are required to determine if they are pheromones.

A non-surgical uterine lavage technique in large cats intended for treatment of uterine infection-induced infertility.

This paper presents the successful use of a non-surgical, transcervical uterine lavage technique for the treatment of uterine infection-induced infertility in three female large cats. We developed a non-surgical uterine lavage technique, which allowed repeated flushing of the uterine lumen and installation of therapeutic antibiotics. The entire procedure was performed under general anaesthesia (duration of anesthesia ranged from 40 to 70 min). It was successfully applied in a Sumatran tiger (Panthera tigris sumatrae), a Corbett tiger (Panthera tigris corbetti) and an Amur leopard (Panthera pardus orientalis). The tigers were treated only once, whereas the leopard received four uterine treatments, due to re-infection after mating. Decisions to conduct uterine treatments were based on detection of uterine fluid during previous transrectal ultrasound examinations. The catheter was guided into the vagina, with the aid of an endoscope, passing the urethra, and then into the uterus, with the aid of transrectal ultrasonography. Both uterine horns were separately flushed with approximately 300 mL of cell medium M199, followed by an antibiotic infusion. Upon ultrasonographic re-examination, the topical uterine treatments resulted in an apparent decline in the inflammatory and/or degenerative processes. The Corbett tiger had the most severe uterine alterations, in addition to an aseptic pyometra. As a result, she was treated 1 month prior to ovariohysterectomy (in order to reduce the surgical risk). The Sumatran tiger was artificially inseminated twice after hormone-induced estrus, and the Amur leopard expressed a spontaneous estrus and re-initiated mating behaviour.

Cryopreservation of captive roe deer (Capreolus capreolus) semen.

To address the need to preserve current genetic diversity before it is lost forever; further studies to adapt assisted reproductive technologies to various endangered species are needed, among other things. Roe deer (Capreolus capreolus), an over abundant wild deer, can serve as model species to develop or improve sperm cryopreservation of threatened or endangered deer species. The aim of this study was to compare the ability of three diluents (Berliner Cryomedium [BC]; Tris, citric acid, glucose [TCG]; TES, Tris, glucose) to support chilling, cryopreservation (with 5% glycerol; G) and postthaw incubation (at 22 °C and 37 °C) of roe deer spermatozoa collected by electroejaculation. Berliner Cryomedium was the diluent that better preserved roe deer spermatozoa during refrigeration, able to maintain motility for at least 14 days, longer than the other extenders. BC + G was the extender of choice for cryopreservation, showing higher viability compared with TCG + G (66.7 ± 3.4 vs. 54.5 ± 6.5; P < 0.05) and higher level of acrosome integrity compared with TES, Tris, glucose + G (79.4 ± 3.4 vs. 67.9 ± 5.0; P < 0.05). Maintaining the samples at 22 °C after thawing presented higher values in various parameters compared with 37 °C. The knowledge gained through this study can potentially act as a preliminary step toward development of new protocols to help increase the reproductive success of biologically similar, yet endangered, wild species.

Seasonal variation in expression and localization of testicular transforming growth factors TGF-{beta}1 and TGF-{beta}3 corresponds with spermatogenic activity in roe deer.

Adult roe deer males show hormonally controlled seasonal cycles of testicular growth and involution. Mediation of endocrine signals likely requires variable production of testicular growth factors for regulation of testis function. Here we studied the expression pattern of transforming growth factors (TGFs) beta1 and beta3. Total RNA from testis parenchyma was extracted monthly and analysed using quantitative reverse transcriptase PCR. The localization of mRNAs was determined by in situ hybridization, and corresponding proteins were visualized immunohistochemically. Both factors showed different expression levels and different seasonal expression patterns. The TGF-beta1 mRNA content was up to 45 times higher than that of TGF-beta3. Compared with its lowest level in May, TGF-beta1 expression was slightly enhanced during pre-rut (June/July). TGF-beta3 expression increased 5-fold from April to June/July and decreased thereafter to its low in December. This corresponded with changing numbers of spermatocytes and round spermatids, in which both TGF-beta3 mRNA and the protein were mainly localized. The TGF-beta1 mRNA was found in interstitial cells, mainly during the non-breeding season, but also in spermatocytes and spermatids during activated spermatogenesis. The translation product was localized in few spermatogenic cells only. The results suggest that TGF-beta1 and -beta3 are important in regulating seasonal spermatogenesis of roe deer with diverse functions affecting interstitial and spermatogenic cells.

Ultrasonographic assessment and ultrasound-guided biopsy of the retropharyngeal lymph nodes in Asian elephants (Elephas maximus).

Endotheliotropic herpesvirus causes a fatal disease in young Asian elephants, but there are no methods for identifying latent carriers of the virus. During the postmortem study of one female African elephant and three male and two female Asian elephants, a lymph node located bilaterally caudoventral to the parotid gland, approximately 1.5 to 5 cm below the skin, was identified as suitable for transcutaneous ultrasound-guided biopsy. An ultrasonographic assessment and two biopsies were performed on 39 Asian elephants, and these lymph nodes were classified ultrasonographically as active, inactive or chronically active. The calculated mean (se) volume of 10 active lymph nodes was 17.4 (6.9) cm(3), and that of three chronically active lymph nodes was 10.6 (1.0) cm(3), whereas the mean volume of 17 inactive lymph nodes was 3.1 (0.6) cm(3). The presence of lymph node tissue in samples obtained by ultrasound-guided biopsy from three animals that were maintained under conditions that allowed for additional sampling was confirmed histologically. The dna extracted from the lymphoid tissue and the whole blood of all the elephants was negative for endotheliotropic herpesvirus by PCR.

A variant of the endotheliotropic herpesvirus in Asian elephants (Elephas maximus) in European zoos.

Newly discovered, lethal elephant endotheliotropic herpesviruses (EEHV) have been identified in both Asian (Elephas maximus) and African (Loxodonta africana) elephants. Carried by otherwise healthy African elephants they can be fatal mainly for young Asian elephants. Since zoos often harbour both elephant species, we conducted a survey on the presence of EEHV in Asian elephants from 12 European zoos, 3 circuses and 1 Israeli zoo. Here, we demonstrate that all EEHV that have affected Asian elephants so far belong to the EEHV1 group. We also describe the detection and the partial sequencing of an endotheliotropic herpesvirus variant (named EEHV1b) in Asian elephants, being either an EEHV endogenous to Asian elephants or indicating different sources (African elephants) of infection.

Histological organization of roe deer testis throughout the seasonal cycle: variable and constant components of tubular and interstitial compartment.

Seasonally regulated breeding in roe deer, Capreolus capreolus, is associated with significant changes in testis mass, structure and function. This study has quantified seasonal changes of morphometric parameters and cellular composition in roe deer testis parenchyma. Tissue samples were collected bimonthly during a complete annual cycle. Morphometric parameters of seminiferous tubules were measured and the number of different cell types was counted using a computer-aided image-analyzing system. A scheme of eight tubular epithelium stages for active spermatogenesis was devised according to the spermatid development. Stage I is characterized by the occurrence of new round spermatids, stage IV by spermiation and stage VIII by the meiotic division of spermatocytes. The average diameter of seminiferous tubules varied between 88.4+/-3.6 micro m (February) and 216.8+/-9.2 micro m (June). Also numbers of spermatogonia, spermatocytes and spermatids per tubule cross-section showed considerable seasonal changes. In December and February the germinative epithelium mainly consists of Sertoli cells and spermatogonia. In February, the first differentiated spermatogonia enter meiosis, and in April even spermatids occasionally occur, which reach their highest numbers during the rut in August. Both the expansion and the proportion of tubular and interstitial compartment change seasonally and result in differing cell densities. Assuming numerically constant populations of Sertoli cells and interstitial cells during the entire year, the hypothetical cell numbers per mm(2) of the tubular and interstitial areas were calculated for the seasonally variable total areas of tissue cross-sections. The concordance of these theoretical values with measured cell densities provided evidence that the total numbers of Sertoli cells, as well as interstitial cells, remain really constant throughout the seasonal cycle. The exact quantification of variable and constant components provides basic data for characterization of cell type and stage-specific processes of spermatogenesis.