Measurement of Alkaline Phosphatase in Canine Seminal Plasma – An Update

In dogs, diagnosis of incomplete ejaculation and azoospermia can be made by measuring the activity of the enzyme alkaline phosphatase (AP) in seminal plasma. However, even though upper cut‐off value of 5000 IU / l is given in the literature, results by different assays may vary considerably. Furthermore, no data exist concerning the stability of the enzyme during storage of frozen seminal plasma, and no recommendations for pre‐analytic dilutions can be found. During the present study, we compared results from a conventional large scale wet chemistry analyzer to a widely used dry chemistry point of care system (POC) and established a best practice for pre‐analytical dilutions. Furthermore, stability of enzyme activities in seminal plasma during storage at ?18°C for 24 h was evaluated. The average activity of AP in the 2nd fraction of normal ejaculates measured by Reflotron® was 107 328 IU / l. After 24 h of frozen storage, activities did not differ significantly (96 844 IU / l, p > 0.05). Fresh and frozen samples were analysed in parallel by the POC and conventional chemistry analyser, and the results compared that did not reveal a significant difference (p > 0.05). A dilution of seminal plasma with physiologic saline 1 : 100 prior to analysis was sufficient for the qualitative information whether AP activity is below or above 5000 IU / l. Present data show that AP measurement by a POC dry chemistry system is sufficiently accurate in diluted seminal plasma for the diagnosis of azoospermia and that seminal plasma can be stored frozen for 24 h before analysis.     

羊驼染色体的研究

对于动物染色体的研究，在国内外有许多学者做了大量的工作，但对于羊驼染色体的研究甚少，仅Taylor、Vidal-Rioja及Bianchi报道羊驼正常二倍体染色体数为2n=74。此外，Wilker，Katrin报道美洲驼有性反转、X染色体单体性及染色体间性等畸变现象，Murray报道羊驼在生理结构及遗传特性等方面与美洲驼非常相似，     

中国羊驼种群MC1R基因的PCR-SSCP分析

探究黑素皮质素受体1(melanocortin 1-receptor,MC1R)基因型与羊驼毛色形成之间的关系,研究采用PCR-SSCP结合DNA测序方法,对60头羊驼个体(51头白色被毛,9头有色被毛)的MC1R基因的多态性进行检测,并分析了MC1R基因位点突变与羊驼不同毛色之间的相关性.结果表明,羊驼MC1R基因存在SSCP多态性,初步判定为AB和AA两种基因型,其中A基因的基因频率为55.0%,B基因的基因频率为45.0%,AB型的基因型频率为90.0%,AA型的基因型频率为10.0%,6个AA基因型均在有色被毛羊驼个体中检出;测序检测显示,AA型羊驼MC1R基因的第801号碱基发生突变,该碱基突变导致编码的氨基酸由异亮氨酸(I)突变为缬氨酸(V).     

羊驼KIT基因exon10-19 cDNA的克隆、表达及生物信息学分析

从羊驼皮肤中提取总RNA,利用RT-PCR技术,扩增了羊驼显性白毛控制基因（KIT）cDNA序列（DQ450844）,并与其它动物相应区域作了同源性比较,结果表明：羊驼KIT基因exon10-19 cDNA长1 044 bp,编码含347个氨基酸残基的蛋白;蛋白质同源性比较显示,羊驼与牛、羊、猪、人、马、猩猩等的同源性大于98%,与鼠的同源性为95%。羊驼aa2编码缬氨酸,而猪、人与牛等动物则编码异亮氨酸,均属于非极性氨基酸;羊驼aa201编码脯氨酸,是非极性氨基酸,而猪、人与牛等动物则编码丝氨酸,是不带电荷的极性氨基酸;羊驼aa344编码精氨酸,而猪、人与牛等动物则编码赖氨酸,均为带正电荷的极性氨基酸。蛋白质二级结构及功能分析结果显示：此二级结构中含有大量的α-螺旋;该蛋白编码肥大细胞/干细胞生长因子,属于酪氨酸激酶受体家族,其蛋白激酶活性位点位于248~260之间。本研究结果将为深入研究KIT基因与羊驼毛色遗传的关系奠定一定的理论基础。     

羊驼催乳素受体基因(PRLR)exon10的克隆与序列分析

通过氰仿／异戊醇法从羊驼血液和组织中提取羊驼基因组DNA，首次扩增出羊驼PRLR基因（Prolactin Receptor，PRLR）exon10序列（GenBank登录号为DQ206831），并与其它动物相应区域作了同源性比较，结果表明：羊驼PRLR基因exon10的开放阅读框为1133bp，包括1046bp的编码区和87bp的拖尾区；同源性比较显示，羊驼PRLR基因exon10的核苷酸序列与哺乳动物（牛、绵羊、猪、狗、兔、大鼠等）的同源性较高，达80％，氨基酸序列的同源性则≥66％，而与鱼类的同源性则较低，仅为40％～45％。     

Effects of Exenatide on Plasma Glucose and Insulin Concentrations in Alpacas

Background: Exenatide is a degradation-resistant glucagon-like peptide 1 agonist used in the treatment of diabetes mellitus. It enhances the insulin response to hyperglycemia. Because of a poor insulin response, adult camelids are susceptible to hyperglycemia from stress, glucose administration, or energy metabolism disorders. Insulin often is administered to decrease plasma glucose concentration, but this approach has disadvantages such as the risk of hypoglycemia. Noninsulin medications targeting the incretin hormone pathway, such as exenatide, are providing alternate treatment options.
Hypothesis/Objectives: Exenatide will decrease plasma glucose and increase insulin concentrations in alpacas.
Animals: Six healthy adult alpacas.
Methods: After food was withheld for 8 hours, alpacas were given, on subsequent days in a randomly determined order, either 0.2 μg/kg of exenatide or similar volume of isotonic saline SC. Blood samples were collected before and 15, 30, 45, 60, 75, 90, 105, and 120 minutes after treatment. A rapid dextrose (0.5 g/kg) injection was given after the time 60 samples. Plasma glucose and insulin concentrations were measured at each time point.
Results: Alpacas had significantly ( P = < .001–.015) lower plasma glucose and higher insulin concentrations for the hyperglycemic period after receiving exenatide than after saline injections. Colic signs were observed in 5 of 6 alpacas treated with exenatide.
Conclusions and Clinical Importance: Exenatide appeared to increase insulin release and decrease plasma glucose concentrations in hyperglycemic alpacas. These findings are similar to findings in humans and could support therapeutic usage of exenatide in alpacas. However, induction of colic may limit practical application.     

Quantification of the DNA Difference,and Separation of X‐ and Y‐Bearing Sperm in Alpacas (Vicugna pacos)

Sperm sexing is an emerging reproductive technology which has been successfully used to produce offspring of a pre‐determined sex in domestic and wildlife species but has yet to be applied to New World camelids. The aims of the present study were to (i) optimize the Hoescht 33342 (H33342) staining concentration for the flow cytometric separation of X and Y chromosome‐bearing alpaca (Vicugna pacos) sperm nuclei, (ii) separate alpaca sperm nuclei into high purity (>90%) populations bearing the X‐ and Y‐chromosome and (iii) determine the DNA difference between X‐ and Y‐bearing sperm in alpacas. Semen was collected from alpacas and sperm nuclei stained with H33342, incubated and analysed using a high‐speed cell sorter (SX‐MoFlo^®). H33342 staining concentrations of 36, 54, 72 or 90 μm did not affect the proportion of correctly oriented sperm nuclei (43.3 ± 3.9, 46.4 ± 3.7, 44.5 ± 4.0 and 51.1 ± 2.5% respectively) nor the speed of sorting (1381 ± 160, 1386 ± 123, 1371 ± 133 and 1379 ± 127 sperm nuclei/s). Sort reanalysis determined high levels of purity for X‐ and Y‐enriched populations (96.6 ± 0.7% and 96.1 ± 1.1% respectively). The DNA difference, based on fluorescence intensity (determined by the SX‐MoFlo^®), was 3.8 ± 0.06%. These data demonstrate for the first time that alpaca sperm nuclei can be separated into high purity populations and the potential for applying sperm sexing technology to New World camelids.     

Unilateral and Bilateral Vasectomy in the Dog: Alkaline Phosphatase as an Indicator of Tubular Patency

Contents The objective of this research was to use a model of unilateral and bilateral occlusion of the ductus deferens in the dog to study the use of alkaline phosphatase (AP) as an indicator of tubular patency. Seven healthy cross bred dogs weighing 10–15 kg BW with normal spermiogram and AP concentrations in semen were used. From each dog, three semen samples were obtained before (intact) and after right (unilateral) and left (bilateral) vasectomy. The AP concentrations were measured in duplicates by a colorimetric method in each of the three fractions (first, second (sperm-rich), third) of each ejaculate. In addition, a macroscopic and microscopic evaluation of each ejaculate was carried out to assure its quality. Data were analysed by least squares analysis of variance using SAS^®. In intact and unilateral vasectomized dogs, 96.6% of AP measured in semen corresponded to the second sperm-rich fraction whereas 1.53 and 1.83% corresponded to the first and third fractions respectively. Total AP concentrations (first and second and third fraction) in vasectomized dogs were lower than in intact animals (19.857 vs 2284.431 ± 4.347 UAL; p < 0.001). AP concentrations were much lower in bilateral than in unilateral vasectomized dogs (142 vs 39.572 ± 4.347 UL, p < 0.001). In summary, AP concentrations in semen can be used as an early indicator of unilateral or bilateral lack of patency of the epididymal and deferent ducts in the dog.     

Effects of parasitic infestation on the productivity of alpacas (Lama pacos)

Summary A trial was carried out in alpacas(Lama pacos) to determine the economic benefit of controlling both external and internal parasites using ivermectin (Ivomec, Merck, Sharpe and Dohme). After four months the treated male alpacas had gained on average 3·1kg more than the untreated males and their fleece weighed 0·36kg more. Treated females gained 1·9kg more than the controls but their fleece weighed 0·03kg less. This resulted in a net financial benefit to the farmer of US$3·54 for each male alpaca and US$1·36 for each female.

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Resumen Se Ilevó a cabo una investigación en alpacas para determinar el beneficio económico del control de parásitos internos y externos, mediante el uso de “Ivomec” (Ivermectin, Merck, Sharpe & Dohme). Después de 4 meses las alpacas machos tratadas habian ganado un promedio de 3·1kg. mas que aquellas que no fueron tratadas y su vellón pesó 0·36kg más. Las alpacas hembras tratadas ganaron 1·9kg mas que los controles, pero su vellón pesó 0·03kg menos. Este tratamiento dio una ganancia neta al ganadero de US$3·54 por cada alpaca macho y $1·36 por cada alpaca hembra.

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Résumé Un essai a été mené sur des alpacas(Lama pacos) pour déterminer le bénéfice économique réalisé par la lutte contre les parasites internes et externes à l'aide de l'ivomec (Ivermectine, Merck, Sharpe & Dohme). Après 4 mois de traitement, le gain de poids pour les males a été d'environ 3,1kg et de 0,36kg pour leur toison par rapport aux non traités. Les femelles avaient gagné 1,9kg en poids vif mais leur toison avait perdu 0,03kg toujours par rapport aux non traités. Cette thérapeutique procure un bénéfice financier net pour le fermier de 3,54$US par male et de 1,36$US par femelle.

     

Diaphragmatic hernia in a two-year-old alpaca (Vicugna pacos)

A 2-yr-old male intact alpaca (Vicugna pacos) was admitted for a 4-day history of anorexia and colic. Five months prior, the alpaca had undergone surgical removal of a duodenal trichophytobezoar and had recovered uneventfully. The alpaca died under anesthesia, and diaphragmatic herniation of the third gastric compartment (C3) was diagnosed at necropsy. A defect was identified in the left dorsal hemidiaphragm accompanied by herniation of 80% of C3 and the aboral portion of the second gastric compartment into the pericardial sac. The smooth margins and dorsal location of the diaphragmatic defect suggested a congenital origin. Diaphragmatic herniation is uncommon in camelids, and only one other case has been reported. Due to the dorsal location of the diaphragmatic defect in this animal, positioning during the previous surgery may have initiated a partial entrapment of gastric compartments, leading to a more complete incarceration between when the animal was discharged and presented again.     

Tuberculosis in alpaca (Lama pacos) on a farm in Ireland. 1. A clinical report

This case report describes tuberculosis (TB) due to infection with Mycobacterium bovis (M. bovis) in alpaca (Lama pacos) on a farm in Ireland. Two severely debilitated alpaca were presented to the University Veterinary Hospital, University College Dublin in November 2004. Bloods were taken, and haematology and biochemistry results were indicative of chronic infection. Radiological examination showed evidence of diffuse granulomatous pneumonia suggestive of tuberculosis. On necropsy there were granulomatous lesions present throughout many body organs including lung, liver, kidney, intestine as well on peritoneum and mesentery. Culture of acid-fast bacilli from lesions led to a diagnosis of tuberculosis due to M. bovis. The use of intradermal skin testing proved inefficient and unreliable for ante mortem diagnosis of tuberculosis in alpaca. Infection due to M. bovis should be considered among the differential diagnoses of debilitating diseases in alpaca, particularly those farmed in areas known to be traditional black spots for tuberculosis in cattle.     

Anthelmintic resistance in a herd of alpacas (Vicugna pacos)

A herd of alpacas was examined because of a history of severe endoparasitism, anemia, hypoproteinemia, and weight loss. Resistance of gastrointestinal nematodes to albendazole, fenbendazole, and doramectin was documented. This report suggests that anthelmintic resistance may be an emerging problem in South American camelids in North America.     

Prostasomes of Canine Seminal Plasma – Zinc‐Binding Ability and Effects on Motility Characteristics and Plasma Membrane Integrity of Spermatozoa

Prostasomes are small lipid membrane‐confined vesicles that are involved in various fertilization‐related processes. The aim of this study was to demonstrate canine seminal plasma prostasomes' ability to bind zinc ions, as well as examining their effects on sperm motility characteristics and plasma membrane integrity during cold storage. Ejaculates, collected from five cross‐bred dogs (n = 50), were subjected to ultracentrifugation followed by gel filtration (GF) on a Superose 6 column. Prostasomes appeared as a single fraction in the elution profile. Transmission electron microscopy (TEM) analysis of canine prostasomes revealed the presence of membrane vesicles with diameters ranging from 20.3 to 301 nm. The zinc‐affinity chromatography on a Chelating Sepharose Fast Flow – Zn^{2 +} showed that from 93 to 100% of the prostasome proteins bind zinc ions (P⁺Zn). SDS‐PAGE revealed that canine P⁺Zn comprised four protein bands, with low molecular weights (10.2–12 kDa). We have also shown a positive effect of prostasomes (p < 0.05), especially variant B (2% of total seminal plasma protein) on canine sperm motility parameters after 2 h storage at 5°C (TMOT%, 44.75 ± 5.18) and PMOT%, 12.42 ± 1.59) and VAP, VSL, VCL, when compared with Control (TMOT%, 7.30 ± 1.41 and PMOT%, 1.70 ± 0.42). Higher percentage of spermatozoa with intact plasma membrane (SYBR/PI dual staining) and intact acrosome (Giemsa stained), after 2 h storage at 5°C, was showed, in variant A (1.5% of total seminal plasma protein) and B, when compared with Control and variant C (2.5% of total seminal plasma protein). The prostasomes' effect on motility and plasma membrane integrity of canine cold‐stored spermatozoa may be related to their ability to bind zinc ions and regulate their availability to the sperm.     

公火鸡外周血浆促黄体激素、睾酮浓度的变化及其与精液品质相关性的研究

8只公火鸡自然光照下饲养,每隔10d收集一次血样和精样,连续5个月(6-10月份)。用放射免疫法测定外周血浆的促黄体激素及睾酮浓度,并用常规方法估测精液品质。结果表明,血浆LH浓度从6月上旬到10月下旬逐步降低,其与白昼长度及湿度呈显著的相关性(r分别为0.553和-0.586)。血浆睾酮浓度的变化趋势与LH相似,血浆睾酮浓度与LH浓度、白昼长度呈显著的相关性(r分别为0.747和0.953)。射精量的变化与白昼长度的变化一致,相关系数为0.655。本实验还表明,LH与射精量呈弱正相关,而与精子密度和死亡精子百分数呈中等强度正相关(r分别为0.368、0.591和0.553)。睾酮浓度与精液品质指标皆呈弱正相关。公火鸡换羽时,血浆LH浓度、射精量,精子密度及死亡精子百分数显著低于非换羽期,睾酮浓度没有发生明显的变化。     

长白公猪血清和精浆中元素含量对精液品质的影响

本试验旨在研究不同精液品质特征长白公猪血清和精浆中元素含量的差异,分析血清和精浆中元素含量对精液品质的影响。基于107头长白公猪的1402次精液品质记录,将公猪群按照精液可利用率划分为3组:低利用率组(利用率<60%,n=21)、中等利用率组(60%≤利用率≤80%,n=27)和高利用率组(利用率>80%,n=59)。采集每头长白公猪的血清和精浆样品,采用电感耦合等离子体质谱法检测血清和精浆中营养元素和毒性元素含量。结果表明:1)低利用率组长白公猪的有效精子数和精子活力显著低于中等利用率和高利用率组(P<0.01),精子畸形率显著高于中等利用率和高利用率组(P<0.01)。2)不同利用率组间长白公猪的血清和精浆元素含量无显著差异(P>0.05);但在血清和精浆元素含量与精液品质参数相关性分析中发现,精浆铅元素含量与精子活力呈显著负相关(P<0.05),与精子畸形率呈显著正相关(P<0.05)。3)对精浆铅含量分组进一步分析发现,精浆铅含量为0μg/L时长白公猪的精子畸形率显著低于精浆铅含量>10.0μg/L时(P<0.05),精子畸形率降低约6.11%。总的来说,长白公猪精浆中毒性元素铅的存在会通过损害精子活力和形态,影响公猪精液品质。     

Treatment of a periradicular lesion in an alpaca (Vicugna pacos)

This case report describes the diagnosis and surgical treatment of a cyst-like periradicular lesion in an alpaca.