A new S. suis serotype 3 infection model in pigs: lack of effect of buprenorphine treatment to reduce distress.

BACKGROUND: Streptoccocus suis (S. suis) is a major porcine pathogen causing meningitis, septicemia, arthritis and endocarditis. These diseases severely impair welfare of pigs. Experimental studies in pigs are important to better understand the pathogenesis and to identify protective antigens, as so far there is no vaccine available protecting against various serotypes (cps). Due to the severity of disease, application of appropriate refinement strategies in experimental S. suis infections is essential to reduce distress imposed on the piglets without jeopardizing the scientific output. The objectives of this study were to evaluate buprenorphine treatment as a refinement measure and serum cortisol levels as a distress read out parameter in a new S. suis cps3 infection model in pigs. RESULTS: Intravenous application of 2 × 108 CFU of S. suis cps3 (sly+, mrp+) to 6-week-old piglets led to severe morbidity in approximately 50% of the animals. Main pathological findings included suppurative meningoencephalitis and arthritis as well as fibrinosuppurative endocarditis. Buprenorphine treatment (0.05 mg/kg every 8 h) did not prevent signs of severe pain, high clinical scores, moderate to severe pathologies or high levels of serum cortisol in single severely affected piglets. Significant differences in the course of leukocytosis, induction of specific antibodies and bactericidal immunity were not recorded between groups with or w/o buprenorphine treatment. Of note, clinically unobtrusive piglets showed serum cortisol levels at 2 and 5 days post infectionem (dpi) comparable to the levels prior to infection with cps3. Cortisol levels in serum were significantly increased in piglets euthanized due to severe disease in comparison to clinically unobtrusive pigs. CONCLUSIONS: Different clinical courses and pathologies are induced after intravenous challenge of piglets with 2 × 108 CFU of this S. suis cps3 strain. The chosen protocol of buprenorphine application does not prevent severe distress in this infection model. Important parameters of the humoral immune response, such as the level of IgM binding to S. suis cps3, do not appear to be affected by buprenorphine treatment. Serum cortisol is a meaningful parameter to measure distress in piglets experimentally infected with S. suis and to evaluate refinement strategies. In this intravenous model, which includes close clinical monitoring and different humane endpoints, clinics and cortisol levels suggest convalescence in surviving piglets within 5 days following experimental infection.

Feasibility of combined upper and lower gastrointestinal endoscopic biopsy in the common marmoset (Callithrix jacchus) to evaluate gastrointestinal diseases.

BACKGROUND: Chronic gastroenteropathies, including gluten sensitivity and marmoset wasting syndrome, frequently occur in captive colonies of common marmosets (Callithrix jacchus). Early identification and diagnosis of affected animals are desirable. Endoscopic examination of the colon in marmosets is described, but the small intestine can harbor significant mucosal lesions not representing those in the colon. Evaluating the small intestine currently requires invasive surgical biopsies due to the small patient size, carrying a risk of severe complications. METHODS: Endoscopic intubation and multisite biopsy of the duodenum/proximal jejunum are demonstrated in 10 marmosets under general anesthesia. RESULTS: Esophagogastroduodenoscopy with colonoscopy efficiently aid in examining the gastrointestinal tract and obtaining an antemortem histologic diagnosis in marmosets with chronic gastrointestinal signs. CONCLUSIONS: This minimally invasive technique is feasible in marmosets. Future investigations into the pathogenesis of chronic gastroenteropathies will benefit from these data, leading to improved animal welfare and better individual and colony health management.

Investigation of Body Development in Growing Holstein Heifers With Special Emphasis on Body Fat Development Using Bioelectrical Impedance Analysis.

This study analyzed skeletal development, body condition, and total body fat development of growing heifers. A total of 144 female primiparous Holstein cattle from four commercial dairy farms with different degrees of stillbirth rates were examined during the rearing period. This included measurements in body condition, fat tissue, metabolic, and endocrine factors. Pelvic measurements and the sacrum height were analyzed to assess skeletal development. The body condition was classified via body condition scoring, bioelectrical impedance analysis (BIA), back fat thickness measurements, and the body mass. For the first time, BIA was used as an appropriate method to evaluate the fat tissue content of cattle throughout the rearing period. This analysis technique can be performed on heifers aged 8-15 months. Throughout that period, the fat content decreased while the skeletal development increased. In addition, high free fatty acid concentrations in serum of the animals with high frame development were found, supporting our hypothesis that stored energy of body fat deposits is used for skeletal growth. Furthermore, we were able to demonstrate complex endocrine relationships between fat metabolism and skeletal growth by using specific markers, such as leptin, insulin growth factor-1 (IGF-1), and estradiol (E2). Food analysis showed high crude protein (CP) levels in the total mixed ration above recommendation for daily protein intake of all farms. However, there was a positive correlation between CP and the body frame measurements in our study. In summary, we established a novel regression formula for BIA analysis ("BIA-Heine") in heifers to evaluate the body composition throughout different ages and physiological stages in the development of heifers. This special formula allows the evaluation of fat tissue without a whole-body analysis and therefore provides an innovative technique for animal welfare support.

[Cortisol concentration before, during and after sham foot trimming in German Holstein cows - the suitability of different matrices]. / Kortisolkonzentration bei Deutsch-Holstein-Kühen vor, während und nach der Klauenbehandlung im Durchtreibestand ­ Eignung verschiedener Matrizes.

OBJECTIVE: The objective of this study was to determine whether changes in cortisol concentration measured in blood serum (KoB) of cows exposed to an acute stressor shows a correlation to cortisol concentrations in saliva (KoS), tears (KoT) and milk (KoM) as well as the concentration of cortisol metabolites in feces (KoK). MATERIALS AND METHODS: In 10 healthy German Holstein cows, sham foot trimming (sKB) including the movement/fixation of the cows through/in a foot trimming chute was used as a model for acute stress. KoB, KoS, KoT, KoM and KoK were measured once a day for 10 days. During sKB, performed on day 4, KoB and KoT were measured at the initiation of foot trimming (minute 0) as well as 15, 25, 30, 40, 50, 60 and 80 minutes later. Additionally, KoK was measured 480, 540, 600 and 660 minutes after the start of sKB. RESULTS: KoB and KoT increased during sKB and reached a maximum at 60 minutes followed by a decrease. KoK increased after sKB and reached a maximum at 660 minutes. There was a significant correlation between KoB and KoT, KoK and KoB and a trend towards a significant correlation between KoK and KoT during sKB. KoB and KoT were significantly correlated (area under the curve, minute 0-10, p = 0.04). KoB decreased significantly from day 1 to day 4 (p < 0.01). On day, 5 KoB (p = 0.03) and KoK (p < 0.01) were significantly higher. KoS and KoT served as good proxies for KoB throughout the study, and KoK and KoB exhibited similar profiles. There were several differences between the profiles of KoM and KoB. During the 10-day measurement period, a significant positive correlation was detected between KoB and KoS (p = 0.002) as well as between KoB and KoT (p = 0.002). CONCLUSIONS AND CLINICAL RELEVANCE: The collection of tear fluid and saliva for determination of cortisol concentrations are non-invasive alternatives to blood sampling. Calm handling of cows may reduce their stress reaction and thus improve animal welfare.

Chemical cues of female fertility states in a non-human primate.

An increasing number of studies suggest that olfaction is important for communication throughout the order of primates. Callitrichids, in particular, have well-developed olfactory systems and use anogenital glands to produce scent marks. Behavioural studies have shown that male common marmosets (Callithrix jacchus) distinguish between odours from the peri-ovulatory and luteal phase of females. However, large gaps remain in understanding the chemical underpinnings of olfactory cues. To investigate whether chemical cues vary with female fertility and reproductive quality, our study combined behavioural bioassays with chemical analyses of the anogenital odours of female common marmosets using gas chromatography-mass spectrometry. We found that cycle states, age and parity have an impact on chemical profiles and further identified affected chemical substances. Our results confirm and expand on previous behavioural evidence for cues of fertility. Our results indicate that cycle-related substances likely act as chemical cues. Males could use such olfactory fertility cues to optimize their mating effort and thereby increase their paternity certainty. This certainty could enhance paternal care for their infants. The results of our study open a promising avenue to find the metabolic pathways from which chemical cues of fertility arise and to unravel their importance during primate evolution in future comparative studies.

Different methods for volatile sampling in mammals.

Previous studies showed that olfactory cues are important for mammalian communication. However, many specific compounds that convey information between conspecifics are still unknown. To understand mechanisms and functions of olfactory cues, olfactory signals such as volatile compounds emitted from individuals need to be assessed. Sampling of animals with and without scent glands was typically conducted using cotton swabs rubbed over the skin or fur and analysed by gas chromatography-mass spectrometry (GC-MS). However, this method has various drawbacks, including a high level of contaminations. Thus, we adapted two methods of volatile sampling from other research fields and compared them to sampling with cotton swabs. To do so we assessed the body odor of common marmosets (Callithrix jacchus) using cotton swabs, thermal desorption (TD) tubes and, alternatively, a mobile GC-MS device containing a thermal desorption trap. Overall, TD tubes comprised most compounds (N = 113), with half of those compounds being volatile (N = 52). The mobile GC-MS captured the fewest compounds (N = 35), of which all were volatile. Cotton swabs contained an intermediate number of compounds (N = 55), but very few volatiles (N = 10). Almost all compounds found with the mobile GC-MS were also captured with TD tubes (94%). Hence, we recommend TD tubes for state of the art sampling of body odor of mammals or other vertebrates, particularly for field studies, as they can be easily transported, stored and analysed with high performance instruments in the lab. Nevertheless, cotton swabs capture compounds which still may contribute to the body odor, e.g. after bacterial fermentation, while profiles from mobile GC-MS include only the most abundant volatiles of the body odor.

Simultaneous detection of three sex steroid hormone classes using a novel yeast-based biosensor.

A biosensor detecting estrogens, progestogens, and androgens in complex samples and in a single step is described. Three Arxula adeninivorans yeast strains were created, each strain producing a different recombinant human hormone receptor and a different fluorescent reporter protein. These strains were then mixed to create G1212/YRC102-hHR-fluo, the biological component of the biosensor. During incubation with G1212/YRC102-hHR-fluo, hormones present in a sample bind to their target receptor, which leads to the production of a specific fluorescent protein. Three fluorescence scans of the yeast suspension determine which fluorescence protein has been produced, thus revealing which hormone receptor (estrogen, progesterone, and androgen) has been activated by the hormones or hormone mimics present in the sample. The biosensor has similar sensitivities to the existing A. adeninivorans cell-based assays. The detection of the three hormone classes in one single experiment reduces the labor and time required to assay for the three hormone classes. The biosensor was also trialed with animal serum samples for the detection of progestogens, androgens, and estrogens and gave results that correlated well with ELISA analysis in case of progestogens. These results highlight the potential usefulness of the biosensor for comprehensive determination of hormone status in samples from veterinary origin. Biotechnol. Bioeng. 2017;114: 1539-1549. © 2017 Wiley Periodicals, Inc.

Blood pressure as prognostic marker for body condition, cardiovascular, and metabolic diseases in the common marmoset (Callithrix jacchus).

BACKGROUND: The increasing life span of Callithrix jacchus in combination with the occurrence of metabolic and age-dependent diseases requires improved health surveillance for this species. METHODS: The health status of 56 marmosets was studied using a non-invasive blood pressure (BP) device. Age-, weight-, and sex-dependent changes were analyzed. Four animals with striking BP findings had follow-up exams. RESULTS: Physiological and pathological BP values could be defined. BP positively correlated with age and weight, while no effect of sex could be found. Measurement time for female and older animals was shorter than for male and younger individuals. Further analysis of the suspicious patients revealed renal or hepatic diseases and cardiac alterations. CONCLUSION: The description of age and weight influences on BP delivers physiological and pathological values for common marmosets. This may contribute to the understanding of aging process and cardiology in this primate species.

Insulinaemic and glycaemic responses to a second meal of a fibre- or starch-enriched compound feed in healthy horses.

This study aimed to investigate the effect of a second meal of a fibre- or starch-enriched compound feed on glycaemic and insulinaemic responses in healthy horses. In a cross-over design, horses were fed either a starch-enriched compound feed (SCF) or a fibre-enriched compound feed (FCF). On days of blood collection, test diets were fed in the morning (0800 h, first meal) and a second meal was fed 510 min after the first meal was finished (second meal). Significantly higher glycaemic and insulinaemic responses were associated with SCF compared with FCF. Feeding FCF for the second meal yielded similar moderate glycaemic and insulinaemic responses compared with the first meal. Feeding SCF as a second meal yielded significantly reduced glycaemic and insulinaemic responses from the first meal. In practice, evaluating glycaemic and insulinaemic responses of a single meal may not sufficiently describe the overall impact if more than one meal is fed per day.

Seasonal and urban effects on the endocrinology of a wild passerine.

In order to maximize their fitness, organisms in seasonal environments rely on external cues to optimally time their life-history stages. One of the most important zeitgeber to time reproduction is the photoperiod, but further environmental cues are assessed to fine-tune reproduction due to year-to-year variation in environmental conditions. However, in urbanized environments, the pervasive artificial light at night has altered the natural signal of light and darkness. Accordingly, artificial light at night was repeatedly shown to affect avian reproductive physiology and to advance seasonal reproduction in birds. However, these experiments were mainly conducted in the absence of further environmental cues to facilitate the investigation of the mechanisms which are still poorly understood. Here, we investigate whether the endocrine system of free-ranging European blackbirds (Turdus merula) correlates with the amount of artificial light at night along a rural to urban gradient while the birds still encounter complementary environmental cues including seasonal variation in day length and temperature. Testosterone and estrone were assessed as metabolites in fecal samples and corticosterone in blood from mist-netted blackbirds. We demonstrate that seasonal fluctuations in abiotic factors, individual conditions, but also light at night affect the reproductive and stress physiology of wild European blackbirds. Elevated artificial night light intensities were significantly positively correlated with corticosterone and negatively with female estrone levels. No effects of artificial light were found for testosterone levels. Our results suggest that female blackbirds in particular perceive even low levels of artificial light at night as a weak but chronic stressor that interacts with the hypothalamic-pituitary-gonadal axis and leads to a reduced secretion of reproductive hormones. These findings point out that the impacts of light pollution are diverse and we only slowly disentangle its multiple effects on physiology, ecology, and biodiversity.

Relaxins enhance growth of spontaneous murine breast cancers as well as metastatic colonization of the brain.

Relaxins are known for their tissue remodeling capacity which is also a hallmark of cancer progression. However, their role in the latter context is still unclear, particularly in breast cancer. In a mouse model with spontaneously arising breast cancer due to erbB2-overexpression we show that exposure to porcine relaxin results in significantly enhanced tumour growth as compared to control animals. This is accompanied by increased serum concentrations of progesterone and estradiol as well as elevated expression of the respective receptors and the relaxin receptor RXFP1 in the tumour tissue. It is also associated with enhanced infiltration by tumour-associated macrophages which are known to promote tumour progression. Additionally, we show in an ex vivo model of metastatic brain colonization that porcine relaxin as well as human brain-specific relaxin-3 promotes invasion into the brain tissue and enhance interaction of breast cancer cells with the resident brain macrophages, the microglia. Relaxin signaling is mediated via RXFP1, since R 3/I5, a specific agonist of the relaxin-3 receptor RXFP3 in the brain, does not significantly enhance invasion. Taken together, these findings strongly support a role of relaxins in the progression of breast cancer where they foster primary tumour growth as well as metastatic colonization by direct and indirect means.

Antibody selection for immunocytochemical characterization of the male reproductive system in Psittaciformes.

The success of breeding programs is limited by the sparse knowledge about endocrine regulation and biochemical reactions in the psittacine male tract. The immunocytochemical analysis of parrots' testicular tissues provides an insight into their reproductive system but is often hampered by the lack of reliable antibodies. In the present study, we tested a large panel of antibodies raised against steroid receptors, steroidogenic enzymes, relaxin peptides including their receptors, and proliferation markers on paraffin sections of testicular tissue from eight psittacine genera representing three continents. All investigated species displayed the tested markers in somatic and germ cells of testis and epididymis, even though cell distribution was partly heterogenous and in species-specific patterns. The 17ß-hydroxysteroid-dehydrogenase-2, 3ß-hydroxysteroid-dehydrogenase, and smooth muscle actin allowed the cross-species differentiation between active and nonactive gonads. The remaining steroidogenic enzymes, steroid receptors, relaxin peptides, and Ki67 proved to be suitable to define reproductive activity depending on the parrot species. Adapting immunocytochemical methods to different psittacines was successful, though various cellular expression patterns do not allow the transfer of results among different parrot species. However, the availability of a reliable repertory of sexual markers is important to examine reproductive biology of psittacine birds.

The influence of gluten on clinical and immunological status of common marmosets (Callithrix jacchus).

BACKGROUND: Common marmosets (Callithrix jacchus) are susceptible to gastrointestinal diseases. Sensitivity to nutritional elements, for example gluten, has been suggested, but a serological screening has not been performed yet. METHODS: A gluten-containing diet was offered to 24 animals, followed by a gluten-free diet. During these diets, serum IgA antibodies to gliadin (AGA), tissue transglutaminase (tTG), deamidated gliadin (ADGA), and glycoprotein 2 (AGP2A) were determined. Body weight, diarrhea, and other clinical symptoms were recorded. RESULTS: Gluten increased AGA, tTG, and AGP2A concentrations in 13 of 24 animals. A significant decline of AGA and AGP2A was seen on gluten withdrawal. Positive (AGA, tTG) animals presented diarrhea more frequently on gluten-containing diet and showed significantly increased body weight on gluten-free diet compared to negative animals. CONCLUSION: Gluten ingestion caused gastrointestinal symptoms in common marmosets, which disappeared on gluten withdrawal. Considering the immunological response to both diets, gluten sensitivity seems to be most likely.

Luteal insufficiency in bitches as a consequence of an autoimmune response against progesterone?

Shortened estrous cycles, embryonic death, and abortion might be associated with insufficient secretion of progesterone by the canine corpora lutea. The aim of this study was to investigate the concentration of progesterone, prolactin, and relaxin in hypoluteoid and control bitches during pregnancy and the nonpregnant cycle. Moreover, canine antibodies against progesterone were analyzed because of a possible connection between embryonic loss associated with hormone changes related to a hormone antibody response. An enzyme-linked immunosorbent assay (ELISA) was developed and optimized for these purposes. Serum samples from 20 short-cycling and 18 control bitches were analyzed. Animals were assigned to pregnant and nonpregnant groups after ultrasound examination. The results show that the nonpregnant, short-cycling bitches had significantly lower progesterone concentrations than the animals of the control group. In German Shepherd dogs, prolactin concentrations were significantly higher in the hypoluteoid pregnant bitches compared with the control group. Relaxin concentrations did not differ significantly between the pregnant hypoluteoid and pregnant control group at any time of measurement. Moreover, increased levels of IgE antibodies against progesterone were found in the serum of six bitches (five short-cycling bitches, one control animal) out of a total of 38 animals. The results indicated a specific binding of the antibodies to progesterone, but a cross-reaction with estradiol and testosterone might occur. The presented data verify the existence of antibodies against progesterone and suggest that these antibodies might play a role in some cases of unexplained pregnancy failure and shortened cycle length.

Testicular biopsy in psittacine birds (Psittaciformes): comparative evaluation of testicular reproductive status by endoscopic, histologic, and cytologic examination.

Knowledge of the reproductive cycle of male parrots is important for examining the male genital tract and for successful breeding, especially of endangered species. To evaluate different diagnostic methods and criteria concerning the classification of reproductive stages, we examined 20 testicular samples obtained at necropsy in psittacine birds of different species and testicular biopsy samples collected from 9 cockatiels (Nymphicus hollandicus) and 7 rose-ringed parakeets (Psittacula krameri) by endoscopy 4 times over a 12-month period. The testicular reproductive status was assessed histologically and then compared with the macroscopic appearance of the testicles and cytologic results. The histologic examination was nondiagnostic in 19 of 59 testicular biopsy samples. By contrast, the cytologic preparations were diagnostic in 57 of 59 biopsy samples. The results of the cytologic examination coincided with the histologic results in 34 of 38 biopsy samples and 18 of 20 necropsy samples. Macroscopic parameters displayed some differences between reproductive stages but provided an unreliable indication of the reproductive status. These results suggest that microscopic examination of a testicular biopsy sample is a reliable method for evaluating the reproductive status of male parrots and is preferable to the macroscopic evaluation of the testicle. Cytologic examination provides fast preliminary results, even when the histologic preparation is not sufficient for evaluation, but results may be erroneous. Thus, a combination of histologic and cytologic examination is recommended for evaluating testicular reproductive status.

Testicular biopsy in psittacine birds (Psittaciformes): impact of endoscopy and biopsy on health, testicular morphology, and sperm parameters.

Histologic examination of a testicular biopsy sample may be required to evaluate the reproductive status of male psittacine birds. The purpose of this study was to evaluate the viability of testicular sampling from live birds by assessing the impact on the birds' health, testicular integrity, and sperm quality. Testicular biopsy samples were obtained by endoscopy 4 times during 12 months from 9 cockatiels (Nymphicus hollandicus) and 7 rose-ringed parakeets (Psittacula krameri). Only 2 of 16 birds showed testicular cicatrization or divided testicular tissue after a single endoscopy. Further complications, such as damage to the air sacs or bleeding, predominantly occurred in subsequent endoscopies. In both species, endoscopy and testicular biopsy caused only minor or transient effects on sperm production and sperm quality. These results support that a single testicular biopsy is a viable method for evaluating the reproductive status of male psittacine birds.

Prenatal stress programs lipid metabolism enhancing cardiovascular risk in the female F1, F2, and F3 generation in the primate model common marmoset (Callithrix jacchus).

BACKGROUND: Many human diseases are modulated by intrauterine environment, which is called prenatal programming. This study investigated effects of prenatal glucocorticoids on the lipid metabolism of three filial generations of common marmosets. METHODS: Pregnant primates were treated with dexamethasone during pregnancy. Body weight and blood lipid parameters of adult female offspring (F1: n = 5, F2: n = 6, F3: n = 3) were compared with age-related female controls (n = 12). RESULTS: F1, F2, and F3 offspring showed significantly lower percentage of plasma n3 fatty acids than controls. F2 and F3 presented higher cholesterol levels, with significantly more LDL cholesterol, significantly less HDL triglycerides and an enhanced cholesterol/HDL cholesterol ratio. Body weight was not significantly affected. CONCLUSIONS: Prenatal dexamethasone led to higher amounts of cardiovascular risk factors and less protective parameters in female F1-F3 offspring. The intergenerational consequences suggest prenatal programming through epigenetic effects.

Lead optimization of 17ß-HSD1 inhibitors of the (hydroxyphenyl)naphthol sulfonamide type for the treatment of endometriosis.

The reduction of estrone to estradiol, the most potent estrogen in human, is catalyzed by 17ß-hydroxysteroid dehydrogenase type 1 (17ß-HSD1). A promising approach for the treatment of estrogen-dependent diseases is the reduction of intracellular estradiol formation by inhibition of 17ß-HSD1. For the species-specific optimization of the (hydroxyphenyl)naphthols, a combinatorial approach was applied and enhanced by a focused synthesis that resulted in the aromatic-substituted (hydroxyphenyl)naphthol sulfonamides. Rigidification of 12 led to the 4-indolylsulfonamide 30, which is a highly active and selective human 17ß-HSD1 inhibitor, as well as a highly potent and selective inhibitor of 17ß-HSD1 from Callithrix jacchus. It shows no affinity to the estrogen receptors α and ß and good intracellular activity (T47D). Thus, compound 30 shows good properties for further ADMET studies and might be a candidate for the in vivo proof of concept in C. jacchus.

Evaluating the reproductive status of the male budgerigar (Melopsittacus undulatus).

Limited knowledge about male psittacine reproduction reduces the success of breeding programmes. Within the scope of fecundity assessment, classification of male sexual status is essential for effective conservation of the species. The aim of the present study was to investigate the testes of male budgerigars (Melopsittacus undulatus), as psittaciform model species to verify their reproductive status by morphological and immunocytochemical examination. Using light microscopy, gonadal samples were categorized resulting in three reproductive states (active, intermediate, non-active). Calculation of testes weights plus measurement of tubular and interstitial dimensions displayed significant (p ≤ 0.05) differences between all three reproductive stages. Lipids in the testicular tubules, analysed by Sudan black staining and fluorescence microscopy (DAPI(2) mode) were highly present in non-active status. Immunocytochemistry involved two different hydroxysteroid dehydrogenases (HSD), 3ß-HSD and 17ß-HSD-2, as markers for steroidogenesis, as well as steroid receptors for androgens (AR), oestrogen (ER) and progesterone (PR). Both HSDs and AR declined in non-active gonads compared to active and intermediate stages, with a positive signal in germ and somatic cells of testis and epididymis. ER and PR were detected in testicular and epididymal cells, similarly expressed in all three stages. The proliferation rate of germ cells in the testicular tubules, obtained by Ki67, differed significantly in active (38.67%), intermediate (32.40%) and non-active (6.01%) status. According to this morphological study, we have been able to establish markers for the reproductive staging of psittacine testes. This knowledge will be useful to deepen reproductive biology in budgerigars.

[Progesterone and pregnanediol-glucuronid concentrations in saliva, milk and urine of female alpacas and their application in pregnancy diagnosis]. / Trächtigkeitsdiagnostik beim Alpaka mittels Bestimmung von Progesteron und Pregnanediol-glucuronid in Speichel, Milch und Urin.

The objective of the present study was the measurement of the pregnancy associated hormones progesterone (P4) and pregnanediol-glucuronide (PdG) in saliva, milk and urine of alpacas and their potential use in pregnancy diagnosis. Sample of blood, saliva, milk and urine were obtained from 36 female alpacas before mating and throughout the pregnancy. Concentrations of P4 and PdG were determined using an enzyme immunoassay (EIA). Pregnancy was checked by ultrasonography at any sampling time. The milk samples were also tested using a commercial on-farm progesterone kit which was designed for dairy cattle. EIA-Concentrations of P4 in blood, milk and urine and urine PdG concentrations were significantly higher in pregnant than in not pregnant alpacas. There was no difference in concentrations of P4 or PdG in saliva. The accuracy of the progesterone kit was 90% for diagnosis of pregnancy and 69% for non-pregnancy. However, 70% of the false positive results also showed relatively high P4 milk concentrations in the EIA. Values of P4 in blood and PdG in urine are comparable to previous reports in alpacas and therefore can be confirmed as an indicator for pregnancy. Saliva seems unsuitable in pregnancy diagnosis in alpacas, whereas milk seems to be an adequate alternative. The use of milk and urine would simplify the pregnancy diagnosis in alpacas since in contrast to the current methods (e. g. blood progesterone) the owners can take the samples. The avoidance of blood sampling results in a considerable stress reduction for the animals. P4 measurement in milk and PdG measurement in urine are good alternatives in pregnancy diagnosis during the first month of pregnancy, when a trans-abdominal ultrasonographic examination is not yet reliable. However, since high values of P4 and PdG only show the presence of active luteal tissue and therefore are indirect markers of pregnancy the diagnosis should be confirmed using ultrasound later in pregnancy.