Erkki V. S. Koskinen

Fertility of frozen-thawed stallion semen cannot be predicted by the currently used laboratory methods.

The aim of the project was to use current simple and practical laboratory tests and compare results with the foaling rates of mares inseminated with commercially produced frozen semen. In Exp. 1, semen was tested from 27 and in Exp. 2 from 23 stallions; 19 stallions participated in both experiments. The mean number of mares per stallion in both experiments was 37 (min. 7, max. 121). Sperm morphology was assessed and bacterial culture performed once per stallion. In Exp. 1, progressive motility after 0, 1, 2, 3, and 4 h of incubation using light microscopy, motility characteristics measured with an automatic sperm analyzer, plasma membrane integrity using carboxyfluorescein diacetate/propidium iodide (CFDA/PI) staining and light microscopy, plasma membrane integrity using PI staining and a fluorometer, plasma membrane integrity using a resazurin reduction test, and sperm concentration were evaluated. In Exp. 2, the same tests as in Exp. 1 and a hypo-osmotic swelling test (HOST) using both light microscopy and a fluorometer were performed immediately after thawing and after a 3-h incubation. Statistical analysis was done separately to all stallions and to those having ≥ 20 mares; in addition, stallions with foaling rates < 60 or ≥ 60% were compared. In Exp. 1, progressive motility for all stallions after a 2 – 4-h incubation correlated with the foaling rate (correlation coefficients 0.39 – 0.51), (p < 0.05). In stallions with > 20 mares, the artificial insemination dose showed a correlation coefficient of -0.58 (p < 0.05). In Exp. 2, the HOST immediately after thawing showed a negative correlation with foaling rate (p < 0.05). No single test was consistently reliable for predicting the fertilizing capacity of semen, since the 2 experiments yielded conflicting results, although the same stallions sometimes participated in both. This shows the difficulty of frozen semen quality control in commercially produced stallion semen, and on the other hand, the difficulty of conducting fertility trials in horses.

A novel automated fluorometric assay to evaluate sperm viability and fertility in dairy bulls

The artificial insemination (AI) industry is in need of an objective and rapid, but inexpensive method to evaluate frozen thawed bull semen ejaculates. This study presents a new fluorescence method that uses an automatized fluorometer and fluorophore stain propidium iodide that stains only those cells with damaged membranes. The fluorescence of the semen sample and the totally killed subsample were measured simultaneously, and viability was calculated. Every semen batch was analyzed before use in AI. For fertility evaluation, the nonreturn rates (NR%) obtained from 92,120 inseminations with the analyzed batches were recorded from 166 bulls (436 batches). This study confirms a 3.9% better NR% for the Finnish Holstein-Friesian breed than for Finnish Ayrshire. There was a clear seasonality in NR%: it differed (5.3%) significantly, being best in summer to autumn (June to October) and lowest in winter (January to March). The fluorometer method was fast and easy. The correlation between the total number of viable spermatozoa in an insemination dose and field fertility was low but significant (r = 0.051, P = 0.016), suggesting that the plasma membrane integrity evaluation can serve as a cost-beneficial quality control method of frozen-thawed semen at bull stations.

QUALITY OF STALLION SEMEN OBTAINED BY A NEW SEMEN COLLECTION PHANTOM (EQUIDAME®) VERSUS A MISSOURI® ARTIFICIAL VAGINA

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 x 10(6) vs 86.2 +/- 8.1 x 10(6) 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.

Allogeneic Transplantation in Low-Grade Malignant Bone Tumours: A New Operative Technique to Avoid Amputation

A massive allografting of deep-frozen cadaver bone or hemi-joint was performed in 11 patients with tumours which were diagnosed as aggressive, low-grade malignancies. One patient died from a pre-existing hepatic insufficiency. The other 10 patients have been followed up from 1 to 8 years. These patients received two bone grafts and eight hemi-joint grafts around the knee joint. The operative procedure consisted of fixation of the graft by stable osteosynthesis, surrounding the graft-host junction with an autogeneic iliac chips cuff and, in cases of hemi-joint grafts, reconstruction of the ligaments. X-ray, scintigraphy and biopsy were used to judge the incorporation of the graft. The patients had to be prepared to face a considerable morbidity with long non-weight-bearing periods and possibly further operations. A full restitution of function was achieved in the bone graft cases. The patients with hemi-joint grafts were also able to retain their limb with a good or satisfactory function. No metastases or recurrences were experienced. The allograft procedure appears to be useful for younger adults, whose long life expectancy makes this operation justifiable, although the fate of cartilage still is unpredictable.

Histological changes in clinical half-joint allograft replacements.

Fourteen biopsies of cartilage and subchondral bone were taken from nine half-joint knee allografts 10 weeks to 8 years after the transplantation. Deep-frozen cadaver grafts were used to replace the defective half-joint after resection for bone tumour. The biopsies revealed a slow substitution of the dead grafted bone and cartilage. Subchondrally, signs of incorporation could be seen from 12 months on. The dead cartilage matrix degraded gradually and slow simultaneous regeneration was observed. Thus, fibrocartilage was seen on the articular surface of the graft at 12 months, chondrocytes were present at 18 months, but islands of hyaline cartilage were not seen earlier than 7 years after the transplantation. In accordance with the earlier clinical findings, the histological signs of rejection were minimal. The long-term result of half-joint allograft transplantation is obviously dependent on the cartilage component.

Acute BRSV infection in young AI bulls: effect on sperm quality.

Bovine respiratory syncytial virus (BRSV) infection is an important part of the calf pneumonia complex, occasionally affecting even adult cattle. However, the pathogenicity of BRSV in animals older than 6 months is often neglected. Finland is free of many contagious diseases in farm animals, and this gives a good opportunity to study the effects of specific pathogens on bovine reproduction. This report describes the deteriorating effects of BRSV epizootics on sperm morphology and fertility of young dairy bulls (n = 79) at a bull station. More than half of the young bulls had a clinical respiratory disease caused by BRSV during their quarantine when they were 6 months old. Four of seven subsequent quarantine groups were affected. Six months later, when these seropositive bulls (n = 54) came into semen production, they had poorer sperm morphology, and the proportion of normal spermatozoa was 74.1% in BRSV-seropositive animals compared with 81.2% in seronegative bulls (n = 25) (p = 0.035). Field fertility was also slightly affected, the 60-day non-return rates were 75.2% and 76.8% for BRSV seropositive and seronegative bulls respectively (p = 0.014). Potential reasons for lowered sperm quality are discussed here.

Human growth hormone in bone regeneration of non-healing fractures.

Twenty adult patients admitted to hospital because of delayed union or non-union of long-bone fractures (including 15 tibial shaft fractures or associated bone lesions with a poor prognosis of consolidation) were treated with human growth hormone (HGH) and compared with a series of eight similar control patients. Each patient in the treatment group was given 16 i.u. HGH ~ every second day for five weeks in addition to conventional orthopaedic therapy. The progress of bone repair was followed by repeated clinical and conventional X-ray examination. The degree of instability of the fracture was evaluated by measuring from two superimposed X-rays the sum of the medial and lateral deflections produced by an applied weight. Bony union ensued in all 20 eases of the hormone-treated series. The time required for stabilization was 8 weeks or less in 12 cases (60% of the series) and 12 weeks or less in 17 eases (85%) (Fig. 1). No excessive callus formation was observed. The degree of instability, as a percentage of the initial value, seemed to decrease linearly with time, as a percentage of the full consolidation time. However, a curved part at the beginning of the graph suggests that the consolidation process started slowly over one or two weeks. Determination of serum HGH, serum calcium and phosphorus, urinary calcium and phosphorus excretion, serum and urine hydroxyproline and the serum calcium x phosphorus product showed that most of these parameters increased for three weeks after the commencement of HGH treatment. The values then remained constant or decreased up to five weeks. The basal HGH level in the serum of the patients and the controls was strikingly low in most instances, with a median of 0.6gg/1 compared with a normal fasting value of 2.5~g/1 (SD 1.5) reported in the literature. A negative correlation (r =

Restoration of Blood Flow in Severely Injured Limbs By Direct Surgical Techniques

In severe orthopaedic trauma with arrest of blood How in the main artery, the chances of restoring the circulation by active reconstruction have to be clinically weighed. If they are considered positive, then concurrent fracture is no contraindication. The decision will be mainly influenced by the presence of other injuries, e.g., involving the thorax or brain, and of hypovolaemic shock attendant on multiple injury. The different types of arterial lesion occurring, either alone or accompanied by skeletal trauma, have been considered in respect of their surgical treatment in previous reports (2, 3, 4, 8), which also deal with their aetiology and trauma mechanism and with the prognosis. Here, in particular, a methodological review shall be presented, including a report on clinical late results obtained in cases of patients who had sustained extremity trauma involving simultaneous arterial injury and fracture of the bone or dislocation of a joint.