Phylogenetic analysis of SRLV sequences from an arthritic sheep outbreak demonstrates the introduction of CAEV-like viruses among Spanish sheep

Small ruminant lentiviruses (SRLVs) cause different clinical forms of disease in sheep and goats. So far in Spain, Maedi visna virus-like (MVV-like) sequences have been found in both species, and the arthritic SRLV disease has never been found in sheep until a recent outbreak. Knowing that arthritis is common in goats, it was of interest to determine if the genetic type of the virus involved in the sheep arthritis outbreak was caprine arthritis encephalitis virus-like (CAEV-like) rather than MVV-like. Alignment and phylogenetic analyses on nucleotide and deduced amino acid sequences from SRLV of this outbreak, allowed a B2 genetic subgroup assignment of these SRLV, compatible with a correspondence between the virus genetic type and the disease form. Furthermore, an isolate was obtained from the arthritic outbreak, its full genome was CAEV-like but the pol integrase region was MVV-like. Although its LTR lacked a U3 repeat sequence and had a deletion in the R region, which has been proposed to reduce viral replication rate, its phenotype in sheep skin fibroblast cultures was rapid/high, thus it appeared to have adapted to sheep cells. This outbreak study represents the first report on CAEV-like genetic findings and complete genome analysis among Spanish small ruminants.     

Changes in Testicular Development, Ultrasonographic and Histological Appearance of the Testis in Buck Kids Immunized Against LHRH Using Recombinant LHRH Fusion Protein

This study was designed to evaluate the effectiveness of recombinant Ovalbumin-LHRL (OL) immunization on changes in testicular size, histological appearance and testosterone production in buck kids. Thirty native buck kids at 18 weeks of age were divided into three groups, control (n = 10), immunization (n = 10) and castration (n = 10) groups. Immunized animals received OL protein generated by recombinant DNA technology. Ultrasonographic and histological examinations of the testes were performed. Animals were slaughtered at 44 weeks of age. Semen and epididymides were evaluated for the presence of sperm cells. Immunized animals generated anti-LHRH antibodies. Testosterone production, testicular and accessory glands development and sperm production were suppressed in the immunized animals (p < 0.01). Semineferous tubule diameters decreased (p < 0.01), basal membrane of the tubule was thickened and hyalinized in immunized kids. Immunization affected ultrasonographic appearance of the testes drastically. While testes of control animals gained their normal ultrasonographic appearance as the age increased, immunized animals had uniform hypoechogenic testicular structure as observed at 18 weeks of age until slaughter. Simultaneous histological and ultrasonographic evaluations indicated that the changes in testicular histology could partly be monitored via ultrasonographic imaging; nevertheless, it is difficult to claim that ultrasonographic image reflects the exact changes in such instances. In conclusion, these results indicate that recombinant OL fusion protein is effective in immunocastration in buck kids and has a potential to be used as an alternative to physical castration. Further researches should be conducted to help assessing reproductive status of testes from ultrasound images.     

Refrigerated Storage of Red Deer Epididymal Spermatozoa in the Epididymis, Diluted and with Vitamin C Supplementation

We have approached the problem of refrigerated storage of epididymal sperm samples from red deer by comparing three options: storing the genital (testicles within the scrotum), diluting the semen in extender or diluting the semen in extender supplemented with an anti-oxidant. Twenty-nine pairs of testes were collected. Spermatozoa from one of each of the pairs were immediately recovered, and diluted to 400 × 10⁶ sperm/ml in Tris-citrate-fructose with 20% egg yolk. Control group was stored as such, and Anti-oxidant group was supplemented with 0.8 m m vitamin C. The remaining epididymides and the diluted samples were stored at 5°C and spermatozoa were analysed at 0, 24, 96 and 192 h for: motility [computer-assisted semen analysis (CASA)], acrosomal integrity, sperm viability (eosine/nigrosine staining), normal tails and chromatin status [sperm chromatin structure assay (SCSA)]. In general, seminal quality decreased with storage time. Vitamin C supported progressive motility better at 24 h (median 42% vs 23% Control and 15% epididymis), reduced the incidence of tail abnormalities and protected chromatin. Storing the semen in the epididymis slowed down motility loss, but slightly increased the occurrence of tail abnormalities and viability was lower at 192 h. However, regarding chromatin status, sperm stored in the epididymis was protected similarly to those diluted in the medium supplemented with vitamin C. Although the differences between the three groups were small, there were some advantages in supplementing the extender with vitamin C. Besides, refrigerating the epididymis may be a good option when immediate processing is not available.     

Reproductive Performance of Rabbit Does Artificially Inseminated via Intravaginal Administration of [des-Gly 10, d-Ala6]-LHRH Ethylamide as Ovulation Inductor

This study was aimed to evaluate the reproductive performance of rabbit does artificially inseminated (AI) with a GnRH analogue [des‐Gly10, d ‐Ala6]‐LHRH. ethylamide to induce ovulation by intravaginal administration, delivered in the seminal dose. In a preliminary experiment, 39 does were divided into three groups (n = 13) that, at the time of AI, received the following ovulation induction treatments: (i) control group: 20 μg of gonadorelin administered intramuscularly; (ii) 25 μg of the GnRH analogue added to the seminal dose; (iii) 30 μg of the GnRH analogue added to the seminal dose. Fertility did not differ between the three groups (control: 80.6%, group 2: 82.8%, group 3: 73.3%). In a second experiment, a large‐scale field trial was conducted to test the use of 25 μg of the GnRH analogue [des‐Gly10, d ‐Ala6]‐LHRH ethylamide delivered in the seminal dose (n = 270) against 20 μg of gonadorelin administered intramuscularly. Fertility was higher (p < 0.05) when ovulation was induced by intravaginal administration of the GnRH agonist (91.1% vs 85.6%). Prolificacy or mortality at birth was never affected by the ovulation induction treatments. In a third experiment, two groups of does [control group (n = 39): ovulation was induced using 20 μg of gonadorelin administered intramuscularly; treatment group (n = 40): ovulation was induced using 25 μg of [(des‐Gly10, d ‐Ala6)‐LHRH ethylamide added to the seminal dose] were inseminated at 42‐day intervals for five successive AI cycles, to test the response to the GnRH agonist after repeated intravaginal administration to the same animals. Fertility and prolificacy were not influenced by the ovulation induction treatment neither there was an interaction between treatment and parity. The last experiment was aimed to determine whether it could be possible to add the GnRH agonist to the semen in the AI Center, just after semen collection and dilution, or it would have to be added in the farm, immediately before AI. Kindling rates did not significantly differ when ovulation was induced by intramuscular injection of gonadorelin (84.5%) or when the GnRH agonist was added to the seminal dose just at the moment (93.8 %) or 24 h before AI (90.4 %), but it was significantly lower when the hormone was added to the semen 32 h before AI (76.3 %). Prolificacy, however, was not influenced by the ovulation induction treatment.     

Development of a genetic manipulation system for Haemophilus parasuis

Haemophilus parasuis is a member of the family Pasteurellaceae and an important respiratory-tract pathogen of swine, which is the etiological agent of Glasser's disease. Because no genetic manipulation system is available for H. parasuis so far, in vivo studies about the role of its genes involved in virulence are unfeasible. Here we demonstrate that H. parasuis has a cyclic AMP (cAMP)-dependent natural transformation system that enables the uptake of DNA in which the ACCGAACTC sequence signal must be present. After improving DNA transformation parameters, such as cAMP and DNA concentration and exposition time of the exogenous DNA, a knockout mutant of H. parasuis defective in the thy gene, encoding the thymidylate synthase enzyme, has been constructed. Data presented in this work open the possibility for the functional analysis of genes involved in the infectious process of this animal pathogen.     

Effect of nucleotides and carob pulp on gut health and performance of weanling piglets

Nucleotides and carob pulp were tested as possible alternatives to antimicrobial growth promoters for newly weaned pigs. Four dietary treatments were used: basal diet, nucleotides, carob pulp and nucleotides + carob pulp. Performance was measured in a group of 72 pigs. Another group of 60 pigs was used for histological measurements of the jejunal mucosa and microbiological measurements by RFLP in ileum and caecum at different post-weaning intervals. Treatment did not affect performance. On the other hand, the morphology parameters of the jejunum were significantly (P < 0.001) affected by day after-weaning. No effects (P > 0.05) of treatment were observed on these parameters. The similarity (intra and inter-group) of the intestinal microbiota decreased with day after-weaning (P < 0.001) and was also affected by dietary treatment (P < 0.05). The nucleotide diets had the highest similarities in the ileum whereas the carob pulp diets had the lowest in the caecum. It is concluded that both nucleotides and carob pulp are able to modulate the changes in microbiota composition after weaning and that whereas nucleotides act in the ileum, carob pulp acts in the caecum.     

Effects of benzoic acid on performance and ecology of gastrointestinal microbiota in weanling piglets

The effects of feeding benzoic acid on weanling pig performance and on the ecology of their gastrointestinal microbiota were evaluated. One hundred and forty-four piglets (4 weeks of age) were used. Half of the piglets were fed a control diet and half were fed the same diet with 0.5% benzoic acid, and their performance was measured for 28 days. At the end of the trial, twelve piglets from each treatment were euthanised and samples of ileal and caecal digesta were collected. The digesta samples were cultured to quantify Lactobacillus spp., Escherichia coli,Enterococcus spp. and Clostridium perfringens and they were also studied by restriction fragment length polymorphism to evaluate the biodiversity degree and the similarity between the profiles of the microbiota. Benzoic acid improved piglets’ performance, and this was associated with a greater ileal microbiota biodiversity.     

Immunocytochemical detection of amylase, carboxypeptidase A, carcinoembryonic antigen and alpha 1-antitrypsin in carcinomas of the exocrine pancreas of the dog.

The immunocytochemical detection of amylase, carboxypeptidase A, alpha 1-antitrypsin, carcinoembryonic antigen (CEA) and keratin in normal canine pancreatic tissue and in carcinomas of the exocrine pancreas of the dog is described. In the normal pancreas, the acinar cells contain amylase, carboxypeptidase and alpha 1-antitrypsin. The pancreatic ducts react with the antikeratin antibody. Twelve out of 14 pancreatic exocrine carcinomas showed immunoreaction with antiamylase antibody, and 10 with anticarboxypeptidase antibody. Five neoplasms reacted with anti-CEA antibody and three with the anti-alpha 1-antitrypsin antibody. It was not possible to find any systematic difference in the immunocytochemical profiles of acinar, tubular and undifferentiated carcinomas. These results indicate that immunocytochemical marking of amylase and carboxypeptidase is of value in the diagnosis of pancreatic neoplasms in the dog, especially if metastasis is the only material available for study and the tumour does not show any diagnostic feature on routine light microscope preparations.