Restoration of spermatogenesis in infertile male chickens after transplantation of cryopreserved testicular cells

1. The aim of this study was to evaluate the ability of frozen-thawed testicular cells transplanted into infertile cocks to restore spermatogenesis and to compare two cryoprotectants (CPA) (dimethylsulfoxide (DMSO) and Biofreeze).

2. A total of 24 infertile White Leghorn (WL) cocks were transplanted with cryopreserved testicular cells from fertile adult donor cocks. Both genetically close and phylogenetically distant chicken breeds were used as donor cocks.

3. Twelve out of 24 WL recipient cocks with cryopreserved testicular cells restored spermatogenesis within 2 months after the transplantation. Six out of 12 recipient cocks with restored spermatogenesis successfully produced progeny expressing the donor phenotype.

4. There was no difference between the CPA in cell viability after thawing or in the number of offspring produced from cryopreserved testicular tissue.

5. The present work represents the first report of production of a donor-derived healthy progeny following frozen-thawed testicular cell transplantation in adult birds. The described results may contribute to preservation of endangered avian species and to maintaining their genetic variability.     

The Ossification of the Pelvic Girdle and Leg Skeleton of the Quail (Coturnix coturnix japonica)

The onset of ossification centres of the pelvic girdle and leg skeleton of the quail in embryos and juvenile birds were studied. Specimens, which were cleared and were stained with Alcian Blue and Alizarin Red S, were examined at the stereomicroscope. The ilium and the pubis began to ossify at the 8th day (E8), whereas the ischium at E9. Perichondral ossification was observed at E6 in the femur, tibia and fibula. A secondary ossification centre was detected in the proximal epiphysis of the tibiotarsus at the 15th post‐hatching day (P15). The patella began to ossify at P30. Regarding the tarsal bones tibiale, pre‐tibiale and fibulare, ossification was observed at the E15, E12 and E16, respectively. The metatarsals II, III, IV ossified at E7, whereas the metatarsal I at E11. The centres of ossification of the 1st phalanges of all digits were observed at E9. At the same day, the ossification centres of the 2nd phalanx of digits II and III as well as the 3rd phalanx of digit III appeared. At E10, ossification was observed in the 2nd phalanx of digit I, in the 3rd phalanx of digit II and in the 2nd and 3rd phalanx of digit IV. In the 4th phalanx of digit III and in the terminal phalanges of digit IV, ossification was observed at E11. The data presented here provide useful baseline information on the normal sequential pattern of ossification in the pelvic girdle and leg skeleton in this species.     

Gene cloning of porcine adiponectin gene from adipose tissue and construction of its eukaryotic expression vector

To clone adiponectin (ADPN) gene from Shaziling porcine adipocyte and construct its eukaryotic expression vector, total RNA was extracted from subcutaneous fatty tissue. One pair of specific primers was designed by Primer 5.0 software according to the sequence of ADPN gene of porcine available in GenBank. The ADPN gene was amplified by PCR from cDNA and cloned into pMD18‐T vector to construct recombinant clonal vector pMD‐ADPN, sequenced and analysed. A recombinant expression plasmid pPICZaA‐ADPN was constructed by subcloning the cloned ADPN gene into the linearized pPICZaA vector. Then, the plasmid pPICZaA‐ADPN was expressed in Pichia pastoris (GS115) by electrotransformation. Western blot and Bradford analysis were used to determine the target protein induced by methanol. Results showed that the genome size of ADPN was 732 bp and encoded 244 amino acid, the nucleotide sequence of ADPN shared 100% identity with that of porcine available in GenBank. Western blot and Bradford analysis showed that the recombinant ADPN was expressed in GS115 correctly and has certain immune activity. The expression level of ADPN was 28.5 μg/ml. In conclusion, the recombinant ADPN could express in eukaryotic expression vector pPICZaA‐ADPN constructed in this study effectively.     

Ketamine as a part of anaesthetic management in a dog with twiddler's syndrome

An 11‐year‐old male German shepherd dog was referred for possible pacemaker implantation. A routine 6‐lead electrocardiogram revealed a third‐degree atrio‐ventricular block with a heart rate of 40 to 45 beats/minute. A transvenous pacemaker implantation procedure was scheduled. The dog was premedicated with 10 µg/kg acepromazine and 5 mg/kg pethidine. A dose of 5 mg/kg ketamine and 0·2 mg/kg diazepam were used for induction and isoflurane in O₂ and a constant rate infusion of ketamine (20 to 30 µg/kg/minute) were administered for maintenance of general anaesthesia. Due to a twiddler's syndrome, the pacemaker had to be repositioned. For the second procedure, the same protocol was employed except for a lower dose of ketamine both for induction (3 mg/kg) and constant rate infusion (10 to 15 µg/kg/minute). Ketamine appeared to be useful for both management of anaesthesia and cardiac pacemaker implantation in the absence of a temporary pacemaker.     

Prepuce and partial penile amputation for treatment of preputial gland neoplasia in two ferrets

Preputial tumours in ferrets are frequently malignant and therefore warrant prompt investigation. As many cases do not respond favourably to surgery, even in combination with radiation therapy, wide surgical resection has been recommended. Such a procedure may necessitate partial or total penile resection but outcomes have thus far not been well described. The current case series describes two ferrets in which surgical resection, including penile amputation, was performed using 10 and 5 mm margins, respectively. In the first case, no recurrence of preputial gland adenocarcinoma was noted for 32 months postsurgery, whereas multiple attempts at surgery and radiation therapy were unsuccessful in the second. These cases suggest that margins of at least 1 cm may help achieve a better outcome. Penile amputation for the treatment of preputial tumours appears to be well tolerated by ferrets, as demonstrated by these cases.     

Total vertebrectomy for stabilisation of chronic spinal lumbar luxation in a paraplegic dog without nociception

An adult male crossbred dog was referred with a history of a road traffic accident that took place 1 month earlier. Neurological examination revealed paraplegia with absent nociception in the pelvic limbs. On epaxial palpation, significant curvature of the anatomical axis of the spine between the third and fourth lumbar vertebrae was observed, with the presence of a bone end almost piercing the dog's skin. Survey radiographs of the lumbar spine revealed severe dislocation between L3 and L4 vertebrae. During surgery, the spinal cord was not visible between the dislocated segments. Because of difficulties in reducing the lumbar luxation during surgery, vertebrectomy and vertebral shortening were performed. After alignment between vertebrae L3 and L5, eight cortical orthopaedic screws and bone cement were used for fixation. After 30 days, the dog started to use a wheelchair and was considered by its owner to have a good quality of life with no evidence of pain. To the authors’ knowledge, this is the first case of severe luxation treated by total vertebrectomy and spine shortening in a dog. This surgery can be considered as an option in the management of severe spine luxation when the spinal cord is physically transected.     

Expression and Hormonal Regulation of Hoxa10 in Canine Uterus During the Peri-implantation Period

Hoxa10, a homeobox gene, is necessary for endometrial receptivity to blastocyst implantation. The aim of this study was to investigate the differential expression of Hoxa10 in canine uterus during early pregnancy and its regulation under different conditions by in situ hybridization. Hoxa10 mRNA was mainly localized in glandular epithelium and myometrium in canine uterus. There was a low level of Hoxa10 expression in the glandular epithelium on days 6, 12 and 17 of pregnancy. On day 20 of pregnancy when embryo implanted, Hoxa10 mRNA was highly expressed in the glandular epithelium surrounding the embryo, but not in the luminal epithelium. The expression of Hoxa10 mRNA gradually declined from day 23 and reached a low level on day 28. In the myometrium, a low level of Hoxa10 mRNA signal was seen on days 6, 12 and 17 of pregnancy and reached a high level on day 20 of pregnancy. During the estrous cycle, a high level of Hoxa10 mRNA expression was seen in the estrous uterus. Either estrogen or progesterone significantly induced the expression of Hoxa10 mRNA in the ovariectomized canine uterus. These results suggest that Hoxa10 expression is closely related to canine embryo implantation and upregulated by estrogen and progesterone.     

Cerebrospinal Fluid Eosinophilia in Dogs

Background: Marked eosinophilic meningitis or meningoencephalomyelitis (EME) is rarely reported in dogs and the cause is usually undetermined. Long-term prognosis for dogs with cerebrospinal fluid (CSF) eosinophilia is variable.
Animals: Twenty-three client-owned dogs.
Methods: Retrospective case series. Dogs with eosinophilic CSF, defined as total nucleated cell count (TNCC) >3 cells/μL with >20% eosinophils, were identified by a computerized search of all dogs having cisternal and/or lumbar CSF analyzed as part of the diagnostic workup between 1992 and 2007.
Results: TNCC in CSF ranged from 4 to 4,740 cells/μL (median 84 cells/μL, reference range ≤3 cells/μL), with 22 to 95% (median 78%) eosinophils in the differential count. An infectious agent was identified on necropsy in 4 of 23 (17%) dogs ( Cryptococcus neoformans [n = 2], Neospora caninum [n = 1], and Baylisascaris procyonis [n = 1]). Each of these dogs had progressive neurologic deterioration. Sixteen dogs had idiopathic EME. Magnetic resonance imaging (MRI) findings were abnormal in 7 of 13 dogs with EME; 2 dogs had focal lesions and 5 dogs had multifocal lesions. Clinical signs in 12 of 16 (75%) dogs with idiopathic EME resolved with prednisone treatment. Three dogs with acute intervertebral disc herniations recovered after decompressive surgery alone.
Conclusions: Idiopathic EME is a common cause of eosinophilic pleocytosis in dogs. MRI findings are variable. Infectious causes of EME were less common and had a poor prognosis.