Identification of Cytokeratins in Bovine Sperm Outer Dense Fibre Fractions

Outer dense fibres (ODF) are important substructures of mammalian sperm tails that are involved in the regulation of sperm motility. In this study, we investigated the identity of several sodium dodecyl sulphate (SDS)‐insoluble ODF proteins. Bovine ODF were purified by separating sperm heads and tails using ultrasound and Percoll^® density gradient centrifugation. Sperm flagella were treated with the detergent cetyltrimethylammonium bromide (CTAB). CTAB‐insoluble material, which reportedly represents the ODF fraction, was collected, and electron microscopy confirmed a highly purified ODF fraction. We found after solubilization of this fraction with SDS that high amounts of insoluble material were retained after centrifugation. SDS‐insoluble material was collected and quantitatively dissolved in 8 M urea. SDS‐gel electrophoresis in the presence of urea revealed polypeptides with apparent molecular masses of approximately 25, 43, and 50 kDa. Subsequent immunoblotting with anti‐cytokeratin antibodies detected two urea‐soluble, SDS‐insoluble proteins with apparent molecular masses of 45 and 66 kDa. The 45‐kDa protein was identified as cytokeratin 19. An antibody reacting with a palette of cytokeratins (CK 1–18 and CK 20), KL1, was the only antibody that reacted with the 66‐kDa polypeptide. We conclude that sperm ODF fractions contain at least one each of type I and type II intermediate filaments. As keratins and intermediate filaments are described as rope‐like structures, we suggest that these intermediate filaments play an important structural or tension‐bearing role in sperm flagella.     

Evaluation of the acute phase response in cloned pigs following a lipopolysaccharide challenge

The objective of this study was to evaluate the acute phase response (APR) in cloned pigs derived from two different cell lines [C1 (n = 2) and C2 (n = 7)] as compared to genetically similar non-cloned pigs (CONT; n = 11) following a lipopolysaccharide (LPS; 25 microg/kg BW) challenge. Pigs were weaned at 21 days of age and maintained in individual pens in the same room until sample collection approximately 1 week later. Blood samples were collected every 30 min for 2 h prior to and 4h after the LPS challenge. Serum samples were analyzed for cortisol, tumor necrosis factor-alpha (TNF-alpha) and interleukin 6 (IL-6). Average gestational length for cloned pigs, 118.8 +/- 0.97 days, was longer (P < 0.005) than that of CONT pigs, 114+/-0.41 days. For serum cortisol, there was a time by group interaction (P < 0.0001) such that the cortisol response was greater in CONT pigs as compared to C2 pigs (P < 0.0001), but not different from C1 pigs (P > 0.74). A time by group interaction (P < 0.0001) was observed for serum TNF-alpha such that the TNF-alpha response was greater in CONT pigs as compared to C2 pigs (P = 0.0002) and tended to be greater (P < 0.06) than C1 pigs. A time by group interaction (P < 0.0001) was also observed for serum IL-6 such that the serum IL-6 response was greater (P < 0.003) in CONT pigs as compared to C2 pigs and there was a trend (P = 0.10) for serum IL-6 to be greater in CONT pigs compared to the C1 pigs. These are the first results to demonstrate that cortisol and proinflammatory cytokine profiles associated with the APR of cloned pigs are altered compared to genetically similar non-cloned pigs. Our results also indicate that the cell line from which clones are derived may dictate the APR. The hormone and cytokine profiles reported herein are a significant contribution towards our understanding, and perhaps our ability to prevent or reduce the incidence of premature deaths in cloned animals and warrants further investigation of the immune system of cloned animals.     

Identification of factors associated with the development of insect bite hypersensitivity in horses in The Netherlands

An Internet-based questionnaire among horse owners was carried out to identify factors affecting the incidence of insect bite hypersensitivity (IBHI) among horses in the Netherlands. Information was obtained for 794 horses of various breeds, but the breed distribution was not representative for the Dutch horse population. Of the horses for which information was available, 56% suffered from IBH and 44% did not. The most common clinical symptoms were pruritus, scaling, and hair loss, occurring mainly at the base of the tail and along the mane. Breed, age, region (and local habitat), stabling, type of bedding in the stable, use of the horse, deworming frequency, and season were associated with significant differences in IBH incidence. Knowledge of the factors influencing the incidence of IBH may make it possible to reduce the number of animals affected and help alleviate symptoms in affected horses.     

The use of a genetic-counselling program by Dutch breeders for four hereditary health problems in boxer dogs

Our group developed a genetic-counselling program for boxer-dog breeders in The Netherlands, using data for cryptorchidism (uni- and/or bilateral), epilepsy, knee-problems (including ligament rupture, fractured or ruptured meniscus, severe osteo-arthrosis of the knee, or a combination of these disorders), and schisis (including cheiloschisis, palatoschisis, or cheilopalatoschisis). We transformed the estimated breeding values (EBVs) into odds ratios (ORs), to enable the breeder to compare the risk for each of the traits for a certain dam-sire combination with the average population risk (set at 1). The goal of the study was to evaluate the use of our genetic-counselling program by Dutch breeders of boxer dogs. We asked breeders of the Dutch Boxer Club to send in an application form for genetic-counselling from June 1 to December 1, 2000. Breeders indicated on this application form three desirable sires for their dam (sire 1, sire 2, sire 3) in random order. On the basis of this information, a counselling report was produced which included ORs for the four diseases in litters of the dam-sire combinations indicated on the application form. Together with the counselling report, the breeders received an evaluation form. We received 129 application forms from 70 breeders, and collected 125 evaluations. Of these evaluations, 96 were informative about the influence of the counselling report on sire choice. The most-important criteria used by breeders to select sires were: the exterior characteristics (60%) and known progeny (52%). Although it was the first time breeders could make use of genetic-counselling, 32% of the breeders indicated that the genetic-counselling played a major role in their sire selection. Breeders expressed little difference in importance for the four genetic traits, but there was a tendency to consider epilepsy more than the others. Breeders hesitated to put long-term population interest above short-term personal interest. Nevertheless, the general conclusion of this study is that breeders acknowledge the value of counselling, especially to reduce the frequency of a threatening disease such as epilepsy.     

Bovine blood neutrophil acyloxyacyl hydrolase (AOAH) activity during endotoxin and coliform mastitis

The dynamics of blood neutrophil acyloxyacyl hydrolase (AOAH) activity, the appearance of endotoxin (lipopolysaccharide, LPS) in blood and the role of blood neutrophil AOAH in the severity of Escherichia coli and endotoxin mastitis were investigated in early postpartum dairy cows experimentally challenged with either endotoxin (n = 6) or E. coli (n = 6). The AOAH activity of blood neutrophils started to decrease significantly at post challenge hours (PCH) 6-24 and 12-24 in the endotoxin and E. coli-challenged groups, respectively; it returned to pre-challenged values at PCH 48 in both endotoxin- and E. coli-challenged groups. The cows were classified as moderate and severe responders according to milk production loss in the non-challenged quarters at PCH 48. There were no severe responders in the endotoxin-challenged group. In the E. coli-challenged group, only 1 severe responder was identified. The pre-challenge neutrophil AOAH activity of the severe responder was approximately 30% lower than that of moderate responders. No LPS was detected in the plasma of endotoxin-challenged cows; neither was it found in the plasma of moderate responders in the E. coli-challenged group at any PCH. However, at PCH 6, a remarkable amount of LPS was detected in the plasma of the severe responder from the E. coli-challenged group. Furthermore, neutrophil AOAH activity was increased by approximately 70% in the severe responder at PCH 6, but it increased by only approximately 15% in moderate responders. This was followed by a decreased neutrophil AOAH activity at PCH 12-24 and 24-72 in moderate and severe responders, respectively; the decreased AOAH activity at those PCH was more pronounced in the severe responder. The pronounced decreased neutrophil AOAH activity during mastitis often coincided with extreme leukopenia, neutropenia and a maximal number of immature neutrophils in the blood. Our results demonstrate that a decrease in neutrophil AOAH activity results in the appearance of LPS in the blood, and low blood neutrophil deacylation activity could be considered as a risk factor for severe clinical coliform mastitis.     

Making a rational choice between ovariectomy and ovariohysterectomy in the dog: a discussion of the benefits of either technique

OBJECTIVE: To determine if ovariectomy (OVE) is a safe alternative to ovariohysterectomy (OVH) for canine gonadectomy. STUDY DESIGN: Literature review. METHODS: An on-line bibliographic search in MEDLINE and PubMed was performed in December 2004, covering the period 1969-2004. Relevant studies were compared and evaluated with regard to study design, surgical technique, and both short-term and long-term follow-up. CONCLUSIONS: OVH is technically more complicated, time consuming, and is probably associated with greater morbidity (larger incision, more intraoperative trauma, increased discomfort) compared with OVE. No significant differences between techniques were observed for incidence of long-term urogenital problems, including endometritis/pyometra and urinary incontinence, making OVE the preferred method of gonadectomy in the healthy bitch. CLINICAL RELEVANCE: Canine OVE can replace OVH as the procedure of choice for routine neutering of healthy female dogs.     

Evaluation of antibody-ELISA and real-time RT-PCR for the diagnosis and profiling of bluetongue virus serotype 8 during the epidemic in Belgium in 2006

In 2006 bluetongue (BT) emerged for the first time in North-Western Europe. Reliable diagnostic tools are essential in controlling BT but data on the diagnostic sensitivity (Se) and specificity (Sp) are often missing. This paper aims to describe and analyse the results obtained with the diagnostics used in Belgium during the 2006 BT crisis. The diagnosis was based on a combination of antibody detection (competitive ELISA, cELISA) and viral RNA detection by real-time RT-PCR (RT-qPCR). The performance of the cELISA as a diagnostic tool was assessed on field results obtained during the epidemic and previous surveillance campaigns. As the infectious status of the animals is unknown during an epidemic, a Bayesian analysis was performed. Both assays were found to be equally specific (RT-qPCR: 98.5%; cELISA: 98.2%) while the diagnostic sensitivity of the RT-qPCR (99.5%) was superior to that of the cELISA (87.8%). The assumption of RT-qPCR as standard of comparison during the bluetongue virus (BTV) epidemic proved valid based on the results of the Bayesian analysis. A ROC analysis of the cELISA, using RT-qPCR as standard of comparison, showed that the cut-off point with the highest accuracy occurred at a percentage negativity of 66, which is markedly higher than the cut-off proposed by the manufacturer. The analysis of the results was further extended to serological and molecular profiling and the possible use of profiling as a rapid epidemiological marker of the BTV in-field situation was assessed. A comparison of the serological profiles obtained before, during and at the end of the Belgian epidemic clearly showed the existence of an intermediate zone which appears soon after BTV (re)enters the population. The appearance or disappearance of this intermediate zone is correlated with virus circulation and provides valuable information, which would be entirely overlooked if only positive and negative results were considered.