Estrus detection in the bitch with a progesterone rapid test (EIA)--a sensible supplement to vaginal cytology

Vaginal smears were taken from 42 bitches and the results were compared with serum levels of progesterone, which were measured by a radioimmunoassay as well as by a qualitative enzyme-immunoassay. With the enzyme-immunoassay it was possible to differentiate low progesterone levels (less than 3.9 ng/ml) and high progesterone levels (greater than 4.2 ng/ml). Timing of insemination or mating of the bitches using both methods showed good pregnancy rates.     

The applicability of a detection method independent of a laboratory for progesterone in the blood plasma of mares (Hygia progesterone test)

A rapid progesterone assay for cow's milk was checked as to whether it was applicable to mares' blood plasma. The "Hygia Progesterone-Test" is an on-farm test which serves for qualitative analysis. It is generally unusable for mares' plasma but sufficiently precise only in cases of larger or smaller progesterone levels. In cases of moderate amounts of progesterone the test is imprecise. The test can be carried out quickly and easily, but the preparation of blood samples takes more time than preparation of milk samples. The test can be recommended for usage in veterinary practice only, but not for animal owners.     

Serum steroid profiles in artificially maturing female Japanese eel,Anguilla japonica

To investigate whether steroid profiles in salmon pituitary homogenate (SPH)-induced artificially maturing Japanese eel, Anguilla japonica, resemble those in other, naturally maturing fishes, the daily changes in 11 steroids were analyzed for a 70-day period (average time needed to reach the maturational phase). Concentrations of most steroids were low and changed on a weekly basis, with maximum values 2–5 days after an SPH injection. Thus, pregnenolone, 17α-hydroxypregnenolone, dehydroepiandrosterone, progesterone, 17α-hydroxyprogesterone, 17α,20 β-dihydroxy-4-pregnen-3-one, androstenedione and estrone levels were barely or not detectable in serum throughout the experimental period, which is largely in keeping with what is known about oogenesis-related steroids in other fishes. In contrast, serum testosterone (T) levels were high, but fluctuated considerably with each SPH injection (about 0.3–8.3 ng/ml). The serum estradiol-17β (E₂) levels increased after SPH injections and gradually rose throughout the experiment, peaking at the end of the experimental period (about 0.2–7.8 ng/ml). Serum levels of 11-ketotestosterone (11-KT) before SPH treatment were higher (approximately 2 ng/ml) than those of the other steroid hormones (less than 0.5 ng/ml). 11-KT levels increased gradually over the experimental period, and, like E₂, levels peaked towards the end of the experimental period (about 15 ng/ml). The observed patterns for T, E₂ and 11-KT are unlike those in other fishes. Furthermore, the consistent elevations in levels of 11-KT, both before and after SPH treatment, are suggestive of an important role for this steroid in controlling oocyte growth.     

Double ovulation in mares. Clinical, hormone analytical and sonographic studies]

108 mares with a total of 135 oestrous cycles were examined. 30% of the mares showed development of double follicles, found by palpation and ultrasonography. Eight cases of double ovulation, four of them synchronous and four asynchronous, were examined closely. These cases of double ovulation showed different growth and different development of the follicular wall. This occurred at the same time in cases of synchronous double ovulation whereas it differed in case of asynchronous double ovulation. The later ovulating follicle was still growing while the first one had already burst. With both forms of double ovulation an equal concentration of progesterone was detected in peripheral blood. On the day of ovulation the rate of progesterone was low (1 ng/ml blood plasma) and it first started rising from the day +1 on. Double follicles matured slower than single follicles. Mares with synchronous double ovulation showed the typical oestrus behavior. This behavior was less distinct after the first ovulation in mares with asynchronous double ovulation. All four mares with asynchronous and two mares with synchronous double ovulation became pregnant. Twins did not occur. A possible relationship between double ovulation and twin pregnancy is discussed.     

The cell count in sheep and goat milk]

The udder health of 404 sheep from 23 flocks and 397 goats from 15 herds in Lower Austria was examined. In order to determine cell levels, the Schalm Test (equivalent to the California mastitis test) and a fluoroscopic "Fossomatic" cell count appliance were employed. The resultant physiological median levels of somatic cell content were established as 71,000 cells/ml for sheep milk and 415,000 cells/ml for goat milk. Significant factors influencing the cell count levels were the milking technique in both species and age in sheep but not in goats. The pathogens most frequently isolated as causes of chronic or latent mastitis were coagulase-positive and -negative cocci.     

Measurement of Resting and Stress-elevated Serum Cortisol in Rainbow Trout Oncorhynchus mykiss in Experimental Net-pens

A commercially available heterogeneous, solid-phase tube enzyme-linked immunoassay (ELISA) was modified and validated for the measurement of serum cortisol in rainbow trout Oncorhynchus mykiss . The assay is accurate and precise. Resting and stress-elevated serum cortisol concentrations were measured in rainbow trout with a sensitivity of 1.5 ng/ml. Fish held in net-pens at a density of 0.4 kg/m³/cm had a resting cortisol level of 16.5 ± 3.8 ng/ml (mean ± SE). At 3 h postdisturbance, serum cortisol levels were not affected by the removal of fish from adjacent net-pens with dip nets or by the use of 200 mg/L tricaine methanesulfonate (MS-222) as an anesthetic for obtaining samples. However, an acute stress (60 s removal from water) elevated serum cortisol levels to 73.7 ± 9.4 ng/ml.     

Hormonal changes accompanying sexual maturation in captive milkfish (Chanos chanos Forsskal)

Steroid hormone profiles accompanying sexual maturation in captive milkfish are described. There were no significant differences in levels of serum estradiol 17- (E₂) and testosterone (T) between immature male and female fish. Mean E₂ levels rose from 0.54±0.11 ng/ml in immature females (Stage 1) to 4.53±1.16 ng/ml in vitellogenic females (Stage 5), while T levels increased from 2.06±0.28 ng/ml to 38.4±9.26 ng/ml. E₂ and T levels were positively correlated to GSI and oocyte diameter. In males, serum T levels increased from 2.5±0.40 ng/ml in immature males to 27.73±5.02 ng/ml in spermiating males. A significantly higher T level was found in males with thick and scantly milt (spermiation index, SPI, 2) compared to males with scanty milt (SPI, 1) or males with copious, fluid milt (SPI, 3).Serum levels of E₂ and T, and the GSI in females rose significantly during the breeding season (April–June 1983). The levels of both steroids dropped below 1 ng/ml in spent females sampled in succeeding months. In immature males, T levels ranged from 1.11 ng/ml to 2.78 ng/ml and rose significantly to 21.52±8.38 ng/ml during the breeding season when GSI peaked. Serum T levels dropped to around 10 ng/ml in the succeeding months when only spent or regressed males were sampled.     

Fish assemblages in European Western Highlands and Western Plains: a type-specific approach to assess ecological quality of running waters

Abstract  After typological pre-classification of 398 calibration sites, fish-based metric models were used to predict the impact of human activities on river quality in European Western Highlands and Western Plains ecoregions. Calibration sites were grouped into six assemblage types and according to their geomorphology; test sites were assigned to their corresponding assemblage type. Five anthropogenic variables were used to describe the impact level of each site and stepwise discriminant analysis was performed to: (i) avoid redundancy between metrics; (ii) examine how selected metrics discriminated impact classes and (iii) predict ecological status for each site of the given fish type. Globally, this approach predicted the impact class correctly for 64% of sites. The difference between observed and predicted impact was more than one class for only 2.5% of the sites. When validating this approach with an independent data set, differences between observed and predicted impact values never exceeded 2 impact classes, but these differences varied in size among countries.     

黄鳝体内睾酮、雌二醇和炔诺酮含量的检测分析

检测并分析了黄鳝体内睾酮、雌二醇和炔诺酮的含量。结果显示，野生和人工饲养的黄鳝，其血清中睾酮、雌二醇的平均值为0．055ng／ml和22．43pg/ml，野生黄鳝中睾酮、雌二醇的含量普遍高于人工饲养的黄鳝，Student′↑s t检验差异显著。在野生及人工饲养的黄鳝体内均未检出炔诺酮。饲养试验的结果表明，如果黄鳝摄食了添加一定浓度炔诺酮的饲料，能检出其体内炔诺酮的残留。     

春季繁殖鮸鱼亲鱼血清中雌二醇、孕酮和睾酮水平变化分析

模拟鮸鱼自然繁殖生境,采用调控水温、光照,并结合注射生物激素,促使鮸鱼在春季达到性成熟,以春季繁殖鮸鱼亲鱼为材料,检测了其血清中雌二醇(E2)、孕酮(P)和睾酮(T)变化情况。结果表明:1~3月,E2、P和T含量很低;从4月份开始E2、P和T含量逐渐上升;5月份E2、P和T的含量急剧上升,最大值分别到达416.8±254.6 pg/ml、1.75±0.41 ng/ml和0.39±0.18 ng/ml;6月份E2、P和T维持较高的含量;多次催产后到7月份,E2、P和T明显下降,均处于较低水平。说明E2、P和T的检测较准确地反映了人工控制条件下春季繁殖鮸鱼亲鱼的生殖生理活动。     

Changes in gonadal hormones during oocyte development in the striped bass,Morone saxatilis

Wild striped bass,Morone saxatilis, were collected from coastal waters and spawning areas to describe the endocrine correlates of oocyte development in non-captive, migratory fish. The fish were classified according to their most advanced oocytes. Serum levels of estradiol (E₂), testosterone (T) and 17-20-dihydroxyprogesterone (DHP) were measured by radioimmunoassay (RIA). Females in the primary growth phase and early secondary growth phase (pre-vitellogenic) had low levels of plasma steroids, ovarian lipid content and gonadosomatic indices (GSIs). Significant increases in E₂, T, ovarian lipid content and GSIs occurred during the vitellogenic phase. Maximum levels of all reproductive parameters were found in prespawning fish sampled in the Hudson River. Mean levels of E₂, T, ovarian lipids and GSIs for these fish were 2.0±0.5 ng/ml, 3.0±0.3 ng/ml, 24±1 mg/g, and 5.6±0.3% (mean±SEM), respectively. In fish induced to spawn with human chorionic gonadotropin (HCG), DHP levels (1.9±0.4 ng/ml) were significantly elevated. Similar levels were found in two fish captured during the spawning season, suggesting that DHP may serve as the maturation-inducing steroid in this species.     

Determination of the urea content of milk in general practice

To determine the urea in milk and thus to get information on the feeding regimen, a method using colour stripes was developped for the extension service in Bavaria. The quantitative results are fairly exact and available within four minutes comparing the colour changes of the stripe with a four-colour-scale. The scale comprises a range from 15 to more than 40 mg urea nitrogen per 100 ml of milk. The resulting colour as a rule lies between two colour shadings. Udder diseases give uncorrect results. The pH values of the milk samples should be situated between 6.6 and 6.9. This test meets the requirements of the extension service in most cases. Thus disturbances of metabolism caused by feeding can be corrected without problems and immediately. The status of lactation seems to have no influence. Most important in this respect are a feed composition adapted to ruminants and a correct ratio between intake of energy and protein.     

Direct and indirect methods of the diagnosis of pregnancy in mares

This review article deals with a critical comparison between the direct clinical diagnosis for the pregnancy of the mare (rectal and in some cases also vaginal exploration) and indirect methods. Both methods are discussed whether they can be seen as a mutual completion or are suitable for their own. The indirect methods for the pregnancy diagnosis include the hormone-analytic tests as progesterone concentration in serum or milk, the biological and immunological measurements for PMSG in the serum, finally the biological and chemical methods for estrogen contents in the urine of the mare. Furthermore the physical methods mainly the ultrasonic detection are discussed.     

Hormonal changes during meiotic maturation and ovulation in the brook trout (Salvelinus fontinalis)

The plasma levels of estradiol-17 (E2), 17, 20-dihydroxy-4-pregnen-3-one (17,20-P) and gonadotropin (GTH) were measured in brook trout (Salvelinus fontinalis) during the period from the end of vitellogenesis to postovulation. Blood samples were taken according to specific stages of maturation, including germinal vesicle breakdown (GVBD) and ovulation. E2 levels were quite high (45 ng/ml) at the end of vitellogenesis (and prior to GVBD) and dropped precipitously by GVBD (2 ng/ml). They remained low through ovulation and postovulation. 17,20-P levels were low prior to GVBD (0.7 ng/ml) and increased dramatically at GVBD (148 ng/ml). The levels of 17,20-P remained high at ovulation (142 ng/ml) and then dropped significantly within 24 h to approximately half of the ovulatory values. They decreased even further by 7 days postovulation. GTH levels rose gradually through GVBD and ovulation from a postvitellogenic level of approximately 3 ng/ml to a 7 day postovulatory value of approximately 10 ng/ml. The overall results; 1) decrease in estradiol prior to GVBD, 2) increase in 17,20-P at GVBD and 3) gradual GTH rise through GVBD and ovulation, are similar to those reported for other salmonids.     

Steroid release from separated theca and granulosa layers of Atlantic salmon (Salmo salar) ovarian follicles: the effects of a purified salmon gonadotrophin preparation

Theca and granulosa layers were removed from ovarian follicles of mature Atlantic salmon (Salmo salar) and were separately incubated under sterile conditions with and without a partially purified salmon gonadotrophin preparation (GTH). Aliquots of the incubation media were removed at intervals and analysed for the steroids 17, 20-dihydroxy-4-pregnen-3-one (1720P), 17-hydroxyprogesterone, progesterone, androstenedione, testosterone and oestradiol. The biosynthesis of C19 and C21 steroids was very largely restricted to the thecal tissue and was markedly stimulated in the presence of GTH. Androstenedione (max 65 ng/ml) and testosterone (max 14 ng/ml) were released from the earliest stages of incubation whereas the release of 17-hydroxyprogesterone (max 51 ng/ml) and progesterone (max 5.5 ng/ml) commenced only after a lengthy induction period. A trace (1.0 ng/ml) of 1720P was produced by the theca in the presence of GTH but oestradiol was not detected. The granulosa preparations released levels of 17-hydroxyprogesterone and androstenedione only marginally above the detection limits (ca 0.7 ng/ml) and there was little stimulation of output with GTH. Oestradiol (max 4 ng/ml) was released only in the presence of GTH. 1720P, progesterone and testosterone were not detected as products of this tissue. These results, together with those derived earlier from incubations of complete follicles support the view that the synthesis of 1720P is essentially a two-cell process in which 17-hydroxyprogesterone produced in the theca is subject to the action of steroid 20-hydroxysteroid dehydrogenase in the granulosa. The temporal pattern of release of steroids in these and earlier experiments is considered in relation to mechanisms of steroid biosynthesis and to their possible roles in oocyte final maturation.     

膜孕激素受体(mPRα)在半滑舌鳎(Cynoglossus semilaevis)卵母细胞成熟过程中的表达特征

通过实时荧光定量PCR方法(qRT-PCR)检测半滑舌鳎(Cynoglossus semilaevis)雌性性成熟不同时期、不同时相卵母细胞中膜孕激素受体(mPRα)的表达,通过qRT-PCR和Western blotting方法检测促性腺激素(HCG)对成熟期半滑舌鳎卵母细胞mPRα表达的影响,同时,采用原位杂交、免疫组化和Western blotting方法研究雌性性成熟半滑舌鳎mPRα mRNA和蛋白在各组织中的表达.对半滑舌鳎性成熟不同时期、不同时相卵母细胞中mPRα mRNA的定量研究结果显示,在成熟时期半滑舌鳎Ⅴ时相卵母细胞的mPRα mRNA表达量最高.HCG对成熟期半滑舌鳎卵母细胞mPRα表达的影响研究结果显示,20 IU/ml HCG比10 IU/ml HCG对成熟时期半滑舌鳎卵母细胞mPRα mRNA和蛋白表达的促进作用更明显,进一步证明mPRα介导孕激素参与了卵母细胞成熟的调控.对mPRα组织表达的定量和定位研究中,Western blotting结果显示,在半滑舌鳎的卵巢、垂体、脑、头肾、肾、肝脏组织中均有mPRα蛋白表达,且在脑、垂体和卵巢中表达量较高,表明mPRα在不同组织中都发挥着一定的作用,且在内分泌相关组织如脑、垂体和卵巢中的作用更明显.原位杂交和免疫组化结果显示,mPRα mRNA和蛋白表达明显定位在卵母细胞膜上,且在其他组织的外周和管腔结构表达.本研究为进一步研究mPRα在膜上的信号转导机制提供了理论基础和形态学依据.     

常规PCR、实时定量PCR检测传染性皮下及造血器官坏死病毒的效果分析

对虾传染性皮下及造血器官坏死病毒(IHHNV)可感染世界各地养殖对虾,给对虾养殖业造成严重经济损失。本实验首次采用实时定量PCR法对广西地区的84份凡纳滨对虾样品进行检测,同时以常规PCR检测作对照。实时定量PCR检测阳性率为79·8%,常规PCR检测阳性率为40·5%,表明广西地区养殖的凡纳滨对虾IHHNV的感染率较高。将二者检测均呈阳性的30份样品扩增产物进行序列分析测序,测序结果通过DNA STAR软件包进行分析,并通过NCBI Blast与GenBank中的序列进行比对。结果证明,测定的是IHHNV序列。30份样品的IHHNV序列很保守,可以分为4种类型,仅有两个碱基的位置发生变异。实时定量PCR检测IHHNV,快速、灵敏、准确,特异性好,可以作为检测对虾感染病毒的有效方法。     

Reproductive cycle in reared male common Japanese conger, Conger myriaster

Annual changes in gonadal histology, gonadosomatic index (GSI), and plasma 11-ketotestosterone (11KT) levels were investigated in reared male common Japanese congers, Conger myriaster. Young fish, 20–30 cm in total length and around 20 g in body weight, caught in November 1996 (group 1) and in September 1999 (group 2) were reared for 3 years in seawater at temperatures ranging from 10 to 20 °C.

In most fish, only spermatogonia occupied the testes for 1 year and a few months after capture. Spermatocytes appeared in February, and both spermatids and spermatozoa appeared in March, in 1998 for group 1 and in 2001 for group 2. Spermiation was observed from May to September and reduction in testis was observed after October in both groups. GSI and plasma 11KT levels changed with progression of spermatogenesis. Although GSI was less than 1.0 and the plasma 11KT level less than 1.0 ng/ml, in the first year in most fish, both increased in the second year of rearing. GSI peaked in June 1998 (5.3±3.0; mean±standard deviation) in group 1 and May 2001 (2.3±1.3) in group 2, and bottomed in October (0.3±0.1) in both groups. Plasma 11KT levels peaked in March 1998 (5.8±1.9 ng/ml) in group 1 and May 2001 (4.4±2.4 ng/ml) in group 2, and bottomed in August in group 1 and September in group 2 (around 0.1 ng/ml in both groups). Spermatogenesis and changes in GSI and plasma 11KT levels were repeated the following year in both groups.

These observations indicate that males have an annual reproductive cycle under rearing conditions. It is possible that wild male common Japanese congers also have multiple spawning seasons in their lives.     

Microsatellite analysis in domesticated and wild Atlantic salmon (Salmo salar L.): allelic diversity and identification of individuals

Samples of wild and domesticated salmon in Norway were genotyped at 12 microsatellite loci to compare allelic variability and investigate the potential of microsatellite markers for identification of individuals. The following loci were amplified: Ssa20, Ssa62NVH, Ssa71NVH, Ssa90NVH, Ssa103NVH, Ssa105NVH, SsaF43; Ssa20.19; Ssa13.37; SsOSL85; Ssa197; Ssa28. All domesticated strain samples displayed reduced variability compared to wild salmon. On average 58% of the allelic richness observed within the four wild stocks were present in the samples taken from domesticated strains. No systematic differences in heterozygosity were observed between samples representing the two groups.

Pairwise genetic distances, as estimated by Fst values and Nei [1978] was 2–8 times higher among domesticated strains than among wild strains. Among the wild stocks, the highest genetic distances were observed between the river Neiden, located in northern Norway, and the other wild stocks located in the southwest of Norway.

Assignment tests indicated that the wild and domesticated salmon could be distinguished with high precision. Less than 4% of domesticated salmon were misassigned as wild salmon, and less than 3% of wild fish were misassigned as domesticated salmon. Fish from individual domesticated strains were identified with similarly high precision. Assignment to wild salmon stocks was less accurate, with the exception of the sample taken from the river Neiden, for which 93% of the individuals were correctly assigned.     

Detection of Renibacterium salmoninarum in salmonid kidney samples: a comparison of results using double-sandwich ELISA and isolation on selective medium

Abstract A total of 1239 kidney samples from four species of salmonid fish, Atlantic salmon, Salmo salar L., brown trout, Salmo trutta L., Arctic charr, Salvelinus alpinus (L.), and rainbow trout, Oncorhynchus mykiss (Walbaum), were screened for Renibacterium salmoninarum using double-sandwich ELISA and bacterial isolation. For bacterial isolation, samples were homogenized, washed, plated onto S-KDM and incubated for 12 weeks. Samples for ELISA were kept frozen until tested. After thawing, 25% homogenates in PBS were heated at 100°C for 15 min in the presence (2.5% v/v) of HemoDe solvent (terpene and butylated hydroxyanisole) and then centrifuged. The supernatant was tested with polyclonal antibodies against whole bacterium in a double-sandwich ELISA. In seven out of 12 groups tested, all samples were negative in both tests. Positive ELISA results occurred in five groups. Renibacterium salmoninarum was isolated on SKDM from samples in four out of these five groups. The ELISA test gave significantly higher numbers of positive samples in three out of the four groups showing positive results in both tests.