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The European mink is an endangered Mustelidae species and thus requires effective conservation measures, although little is known about reproduction in this species. In particular, preimplantation development has not been studied and, therefore, embryonic development and the growth of embryos was documented in the present study for European mink using light and fluorescent microscopy. Embryos develop in the oviducts and then migrate into the uterus on Day 6 post coitum (p.c.) at the morula stage. Embryos expanded as blastocysts from Day 7 until implantation on Day 12 p.c. Based on these findings, the use of embryo transfer for a conservation programme for the European mink was evaluated. Embryos were flushed from European mink resource females and transferred into the uterine horns of recipient hybrid females (honoriks and nohoriks). These hybrids were obtained by mating European polecat males with European mink females and vice versa. A total of 40 embryos was transferred and 20 live kits were born. The rates of pre- and postnatal survival were 50% and 70%, respectively. Both male and female offspring were lighter at birth in the embryo transfer group compared with naturally born controls, but there was no difference at 3 months of age.

A study was performed to test a new semen collection device (Equidame® phantom) that fractionates the ejaculate by comparing the quality of semen obtained by the Equidame® phantom with that obtained by a Missouri® artificial vagina. Semen from 4 Finnhorse stallions was collected 4 times per stallion by both methods. Half of the ejaculate was frozen and the other half extended and loaded into 2 Equitainer® transport containers (24- and 48-h samples). Motility parameters were determined by a Hamilton-Thorn motility analyzer after cooled storage for 24 and 48 h and again after freezing/thawing. Raw and chilled semen samples were cultured and the number of bacterial colonies counted after incubations of 24 and 48 h. After a 24-h incubation the number of colony-forming units (CFU) in raw semen was significantly higher (P<0.01) when collected by the Missouri® artificial vagina than by the Equidame® phantom. After cooled storage, 75% of the semen samples contained no bacteria after an incubation of 24 h, and 69% yielded no growth after 48 h. The sperm-rich fractions (Cup 2) collected by the Equidame® phantom had lower mean volumes (22.1 ± 2.3 mL [± SEM] versus 101.6 ± 9.3 mL) and significantly higher mean sperm concentrations (218.0 ± 25.8 × 10 vs 86.2 ± 8.1 × 10 cells/mL; P<0.05) than the total ejaculates collected by the Missouri® device. The total and progressive motility of chilled and frozen-thawed semen did not differ significantly between collection methods. The Equidame® phantom yielded semen that was of a lower bacteriological colony counts, but had sperm motility similar to that of semen collected with the traditional method by the Missouri® artificial vagina.

The relationship between the human papillomavirus (HPV) and cancer of the upper aerodigestive trace is controversial. There is no doubt that at least some nasal, oral, pharyngeal and laryngeal carcinomas contain HPV DNA. However, the diversity in the reported incidence of HPV in these tumours represents a major problem. In the present study we have examined 30 laryngeal dysplastic lesions for HPV by the polymerase chain reaction, using three different pairs of consensus primers (GP5+/GP6+, MY09/MY11 and CPI/CPII). The single HPV-positive specimen was from a recurrent laryngeal papilloma. The HPV type present could not be determined, but it was not type 6, 11, 16, 18, 30, 31, 33 or 45. Restriction fragment length polymorphy analysis suggested a yet unknown HPV-type in this lesion. This investigation does not support the idea of HPV as an oncogenic cofactor in most laryngeal carcinomas as HPV was present in only 1/30 precancerous laryngeal lesions.

Summary During the period from April, 1965, to October, 1979, 1,106 patients with symptoms or signs of cervical disc disease were operated on using Cloward's technique. 94% of the patients alive filled in a questionnaire forwarded on October, 1980. The study revealed that 81% experienced an immediate effect of the operation, and 63% were in a better condition than preoperatively at the time of the questionnaire. It appeared that the effect of the operation 1 year postoperatively was independent of age and observation time, but the functional result and postoperative working capacity were very significantly better in young patients, but still independent of observation time. The result was independent of the number of primarily inserted grafts, cutting of the posterior spinal ligament or the finding of a free herniated disc. Use of calfbone grafts resulted in more reoperations.

Summary During a fifteen-year period 1,106 patients underwent Cloward's operation due to cervical disc disease. An analysis of the clinical features showed that the diseases had a very complex pattern of symptoms and signs. In young patients a syndrome of herniated disc predominated whereas a clinical picture of “cervical spondylosis” was found more often in the elderly. It was demonstrated that best results were obtained in young patients, patients with slight symptoms and patients with monoradicular symptoms. A short duration of symptoms is essential for a good outcome, and, on the basis of the data presented, operation is recommended 3 months after the onset of symptoms of cervical disc disease, if previous conservative treatment has failed.

Summary Among 1,106 patients with cervical disc disease treated with Cloward's operation over a fifteen-year period, 138 patients had symptoms and signs of spinal cord involvement. The median symptom duration was one year. Nearly all had gait disturbances, 63% had spasticity of the lower extremities and 11% had various degrees of tetraplegia. Three months after surgery 78% of the patients were found to have some symptoms, but only 5% had no symptoms at all. At the time of the questionnaire (average 7.7 years postoperatively) 46% of the patients stated they were still affected by the operation. The duration of preoperative symptoms was of significance to the outcome, but age, sex, and severity of myelopathy were not. It was demonstrated that patients with medullary involvement had a higher annual mortality rate than patients with radicular symptoms, and that this rate depended on the severity of the spinal cord symptoms.

The case histories of 4 patients are presented, who developed an invasive squamous cell carcinoma from pre-existing multiple laryngeal papillomatosis (two juvenile-onset and two adult-onset) during a long latency period. A series of 14 routinely processed, paraffin-embedded biopsies obtained from these patients (including both benign papillomas and carcinomas) were analysed using in situ DNA-hybridization technique with 35S-labelled Human papillomavirus (HPV) DNA probes of HPV types 6, 11, 16, 18 and 30. The lesions were also assessed by indirect immunoperoxidase (IP-PAP) technique to demonstrate the expression of HPV structural proteins. On light microscopy, morphology was consistent with HPV infection in all 14 biopsies. HPV antigen expression could not be demonstrated in any of the papillomas or carcinomas, however. HPV 11 DNA was present in high copy numbers in both the benign and malignant lesions of 2 patients, both presenting with the juvenile-onset disease. The present findings provide support for the role of HPV as an etiological agent in laryngeal squamous cell carcinoma, most probably acting synergistically with chemical or physical carcinogens (one patient received irradiation and 2 others were smokers). It seems clear that an infection by the 'low risk' HPV types 6 and 11 by no means excludes the possibility of developing a laryngeal malignancy, so far ascribed to the 'high risk' type HPV 16 only.

Postmortem submandibular glands from 14 patients with late-onset diabetes mellitus were examined in a histomorphometric study and were compared with 14 control glands. The diabetic glands were larger than the control glands and showed increased volume fractions of the stromal components, including vessels, and proportionally less parenchyma. These findings indicate that the increased total volume present in submandibular glands in lateonset diabetes is mainly to increased volumes of fibrous tissue, fat and vessels.

Summary Among 1,106 patients with cervical disc disease in whom Cloward's operation had been performed over a fifteen-year period, 145 patients were reoperated upon with a new Cloward procedure. In the 1st year after the first operation 3/4 of the patients were operated upon on the previous level, whereas reoperations performed later mainly were done on new levels. The effect of the reoperation was poorer than the effect of the 1st operation. Early reoperations—which were due to: an initially unsatisfactory clinical course, postoperative complications or graft problems—had a poor result, but late reoperations—which were caused by relapses—showed a result corresponding to the first operation. The frequency of relapses leading to a 2nd Cloward operation was 8.8% in the first year falling to 0.2% in the following years of observation.

Summary During a fifteen-year period 1,106 patients with symptoms and signs of cervical disc disease underwent Cloward's operation. Plain x-rays were performed in 94%, but the severity and extension of degenerative changes had no prognostic value. In 91 % of the patients myelography was performed and in recent years mainly metrizamide was used as it was found to be more accurate. The findings on the myelograms were correlated according to age, severity and number of affected disc levels, but were independent of the duration of symptoms. Patients with medullary symptoms were found more often than not to have an anterior indentation into the spinal canal whereas most patients with radicular symptoms had wide cervical root sleeves or lateral compression. The best outcome was found among patients with monosegmental symptoms and signs and one affected disc level on the myelogram underlining that the indication for surgery in cervical disc disease should be based on both clinical and radiological findings.

AIM: To examine conjunctival papillomas for the presence of human papillomavirus (HPV) and koilocytosis. METHODS: Archival paraffin embedded tissue from 55 conjunctival papillomas was analysed for the presence of HPV by polymerase chain reaction and subsequent filter hybridisation. Histological sections of the 55 papillomas were evaluated for the presence of koilocytosis. RESULTS: HPV was present in 48 of 52 (92%) β globin positive papillomas. HPV type 6/11 were found in 40 of 47 investigated papillomas and a double infection with HPV 6/11 and 16 was identified in a single papilloma. In six papillomas the HPV type could not be identified. Koilocytosis was present in 22 of 55 papillomas (40%). CONCLUSION: There is a strong association between HPV and conjunctival papillomas. HPV type 6/11 is the most common HPV type in conjunctival papilloma. The sensitivity of koilocytosis as an indicator of HPV in conjunctival papilloma is low.

The effect of in vitro culture on viability of pre-implantation stage embryos in the farmed European polecat was studied, aimed at developing assisted reproductive technology for conservation of endangered mustelids, particularly the European mink (Mustela lutreola). Embryo storage in culture would enable embryo recovery and transfer in different locations. Ferret (Mustela putorius furo) kits have been produced from embryos that were cultured for 3 days in serum-containing medium (Li et al. 2001 Reproduction 122, 611?618). In our earlier studies, polecat embryos were maintained for 24 h in culture conditions (Lindeberg et al. 2003 Theriogenology 60, 965?970). Fourteen estrous donors were kept in the same cage with a fertile male overnight and sacrificed 3 days after the start of mating for recovery of embryos from the oviducts. Embryos were flushed with Emcare? Complete ultra flushing medium (ICPBio, Auckland, New Zealand), washed twice in it, washed once in Emcare? embryo holding solution and transported in the holding solution at room temperature for 1 h to the laboratory. Embryos of seven donors were pooled and cultured in 30-?L drops of TCM199 + glutamax I (GIBCO?) supplemented with fatty acid-free albumin (FAFBSA, Sigma-Aldrech, Helsinki, Finland) under a cover of paraffin oil (Medicult) for 3 days in a humidified atmosphere (39C) and in 5% of O2. At the end of the culture, the embryos were evaluated and the ones that had developed at least to morula stage were chosen for transfers. The selected embryos were transported at room temperature in Emcare? embryo holding solution for 1 h to the farm where they were surgically transferred under general anesthesia into seven recipients. The recipients had been mated the same way as the donors but with vasectomized males either on the same day as the donors (the first set: 7 donors, 3 recipients) or one day later than the donors (the second set: 7 donors, 4 recipients). Five embryos were cultured a total of 6 days to the blastocyst stage and stained for a count of cell numbers. A total number of 169 one- to 16-cell-stage embryos were recovered. At the end of the 3-day culture period, a total of 139 (139/169, 82%) had developed to morula (56.6%), compact morula (9.8%), early blastocyst (30.3%), or blastocyst stage (3.3%). Of these 139 embryos, a total of 102 were surgically transferred. Five of the 7 recipients delivered one to 5 kits each 43 to 45 days after the mating. Altogether 21 kits were born and the success rate was 21% (21 kits/102 transferred embryos). Cell numbers of the five Day 6 blastocysts varied from 130 to 430. In conclusion, this preliminary trial confirms that polecat embryos can be stored in culture for 3 days. In this study polecat embryos were cultured in 5% oxygen and without addition of serum which resulted in considerably better cell numbers for Day 6 blastocysts than in our earlier studies (90 to 165 cells; Lindeberg et al. 2003 Theriogenology 60, 965?970).

The effect of in vitro culture on viability of pre-implantation stage embryos in the farmed European polecat was studied, aimed at developing assisted reproductive technology for conservation of endangered mustelids, particularly the European mink (Mustela lutreola). Embryo storage in culture would enable embryo recovery and transfer in different locations. Ferret (Mustela putorius furo) kits have been produced from embryos that were cultured for 3 days in serum-containing medium (Li et al. 2001 Reproduction 122, 611?618). In our earlier studies, polecat embryos were maintained for 24 h in culture conditions (Lindeberg et al. 2003 Theriogenology 60, 965?970). Fourteen estrous donors were kept in the same cage with a fertile male overnight and sacrificed 3 days after the start of mating for recovery of embryos from the oviducts. Embryos were flushed with Emcare? Complete ultra flushing medium (ICPBio, Auckland, New Zealand), washed twice in it, washed once in Emcare? embryo holding solution and transported in the holding solution at room temperature for 1 h to the laboratory. Embryos of seven donors were pooled and cultured in 30-?L drops of TCM199 + glutamax I (GIBCO?) supplemented with fatty acid-free albumin (FAFBSA, Sigma-Aldrech, Helsinki, Finland) under a cover of paraffin oil (Medicult) for 3 days in a humidified atmosphere (39C) and in 5% of O2. At the end of the culture, the embryos were evaluated and the ones that had developed at least to morula stage were chosen for transfers. The selected embryos were transported at room temperature in Emcare? embryo holding solution for 1 h to the farm where they were surgically transferred under general anesthesia into seven recipients. The recipients had been mated the same way as the donors but with vasectomized males either on the same day as the donors (the first set: 7 donors, 3 recipients) or one day later than the donors (the second set: 7 donors, 4 recipients). Five embryos were cultured a total of 6 days to the blastocyst stage and stained for a count of cell numbers. A total number of 169 one- to 16-cell-stage embryos were recovered. At the end of the 3-day culture period, a total of 139 (139/169, 82%) had developed to morula (56.6%), compact morula (9.8%), early blastocyst (30.3%), or blastocyst stage (3.3%). Of these 139 embryos, a total of 102 were surgically transferred. Five of the 7 recipients delivered one to 5 kits each 43 to 45 days after the mating. Altogether 21 kits were born and the success rate was 21% (21 kits/102 transferred embryos). Cell numbers of the five Day 6 blastocysts varied from 130 to 430. In conclusion, this preliminary trial confirms that polecat embryos can be stored in culture for 3 days. In this study polecat embryos were cultured in 5% oxygen and without addition of serum which resulted in considerably better cell numbers for Day 6 blastocysts than in our earlier studies (90 to 165 cells; Lindeberg et al. 2003 Theriogenology 60, 965?970).