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.

Uterine insemination with a standard AI dose in a sow pool system.

The effect of uterine AI with a standard dose of spermatozoa on fertility of the sow was studied in a field trial. The trial involved a sow pool system with 440 sows using AI as the primary method of breeding. Sows were twice a day checked for oestrus symptoms by back pressure test in front of a boar on days 3-6 after weaning. When in standing heat, sows were randomly allocated into either a uterine insemination group (UTER, n = 157) or standard AI group (CONT, n = 169) and bred accordingly using 3 billion spermatozoa in 80 ml of extender. In both treatment groups, insemination was repeated once if the sow was still receptive 24 h later. Using pregnancy (farrowed or not) and live-born litter size as the outcome variables, a logistic and linear regression approach, respectively, was taken to study the effect of the following factors: treatment (UTER vs CONT), AI operator, breed, satellite herd preceding weaning, parity, weaning-to-oestrus interval and length of lactation. Overall, live-born litter size was 11.3 +/- 2.9, repeat breeding rate 4.2% and farrowing rate 91.2%. In the UTER group, 93.6% of inseminated sows farrowed, whereas farrowing rate for the CONT group was 88.8% (p = 0.13). Intrauterine insemination with a standard AI dose did not result in a significant improvement in the live-born litter size (11.5 +/- 2.8 for the UTER and 11.1 +/- 3.0 for the CONT sows, respectively, p = 0.13). However, the preceding satellite herd had a highly significant effect on the live-born litter size (12.4 +/- 2.6; 11.1 +/- 2.9; 10.8 +/- 2.9 and 10.9 +/- 2.9 for the four satellite herds, p < 0.01). We conclude that uterine insemination did not have a significant effect on live-born litter size and farrowing rate and we also conclude that satellite herd appears to have a major effect on fertility in a sow pool system.

Non-invasive, label-free cell counting and quantitative analysis of adherent cells using digital holography.

Manual cell counting is time consuming and requires a high degree of skill on behalf of the person performing the count. Here we use a technique that utilizes digital holography, allowing label-free and completely non-invasive cell counting directly in cell culture vessels with adherent viable cells. The images produced can provide both quantitative and qualitative phase information from a single hologram. The recently constructed microscope Holomonitor (Phase Holographic Imaging AB, Lund, Sweden) combines the commonly used phase contrast microscope with digital holography, the latter giving us the possibility of achieving quantitative information on cellular shape, area, confluence and optical thickness. This project aimed at determining the accuracy and repeatability of cell counting measurements using digital holography compared to the conventional manual cell counting method using a haemocytometer. The collected data were also used to determine cell size and cellular optical thickness. The results show that digital holography can be used for non-invasive automatic cell counting as precisely as conventional manual cell counting.

Inhibition of cell proliferation and induction of apoptosis by N(1),N(11)-diethylnorspermine-induced polyamine pool reduction.

Reduction of cellular polyamine pools results in inhibition of cell proliferation and sometimes in induction of cell death. Reduction of cellular polyamine pools can be achieved by several strategies involving all the mechanisms of polyamine homoeostasis, i.e. biosynthesis, catabolism and transport across the cell membrane. In the present paper, we concentrate on results achieved using the polyamine analogue DENSPM (N(1),N(11)-diethylnorspermine) on different cell lines. We discuss polyamine levels in DENSPM-treated cells in relation to effects on cell cycle kinetics and induction of apoptosis. To really understand the role of polyamines in cell cycle regulation and apoptosis, we believe it is now time to go through the vast polyamine literature in a meta-analysis-based manner. This short review does not claim to be such a study, but it is our hope to stimulate such studies in the polyamine field. Such work is especially important from the viewpoint of introducing drugs that affect polyamine homoeostasis in the treatment of various diseases such as cancer.

Porcine field fertility with two different insemination doses and the effect of sperm morphology.

In swine artificial insemination, several dose regimens are applied, ranging from 1.5 x 10(9) to 6.0 x 10(9) spermatozoa per intra-cervical insemination dose. A lower sperm dose is more profitable for artificial insemination centres and offers a more effective use of superior boars. To evaluate fertility, 50 boars were used for a total of 10 773 homospermic first inseminations at a dose of 2 billion spermatozoa. In addition, 96 boars were used at a dose of 3 billion spermatozoa for 34 789 homospermic first inseminations. Fertility was determined by a 60-day non-return rate (NR%) of first inseminations. Litter size was registered by total number of piglets born separately in primiparous and multiparous farrowings. On average, a sow was inseminated 1.5 times. A significant decrease was observed in all three fertility parameters (NR%, litter size of both primiparous and multiparous farrowings) with a dose of 2 billion spermatozoa compared with a dose of 3 billion spermatozoa. The NR% was 75.8% and 84.0% (p < 0.001), the mean litter size of primiparous farrowings 10.1 and 10.7 (p < 0.001) and the mean litter size of multiparous farrowings 11.7 and 12.1 (p < 0.001) for 2 and 3 billion spermatozoa/dose, respectively. The proportion of normal spermatozoa in the sperm morphology analysis correlated significantly with NR% in both insemination regimens: p < 0.001, r = 0.604 and p < 0.05, r = 0.223 for 2 and 3 billion spermatozoa/dose, respectively. These results confirm that quantity can at least partly compensate for poor sperm quality. When the boars with <70% normal spermatozoa in the morphology evaluation were excluded from the data there were no correlation between the sperm morphology and NR%. However, the difference between the NR% and litter size remained statistically significant (p < 0.001) in favour for the bigger insemination dose. In conclusion, a decrease in sperm dose from 3 to 2 billion spermatozoa on commercial farms will severely decrease prolificacy at least under field conditions, where a sow is inseminated an average of 1.5 times/heat, and the semen is typically used within 3 days after collection. We recommend that under commercial circumstances the homospermic semen doses contain no <3 billion spermatozoa/dose.

Impaired semen quality of AI bulls fed with moldy hay: a case report.

The daily quality control of semen at a Finnish artificial insemination (AI) bull station is based on subjective motility and sperm morphology of young bulls entering the semen collection program. Semen quality dropped suddenly in autumn 1998. During 5 consecutive months, the number of rejected ejaculates and discarded frozen semen batches due to poor motility increased, and the number of all forms of abnormal spermatozoa increased. However, for the accepted ejaculates, a 60 day nonretum rate was normal. The summer of 1998 in Finland was rainy, and the hay used in the AI station was visibly moldy. Immunoassay and gas chromatography-mass spectrometry (GC-MS) detected Fusarium mycotoxins HT-2 and T-2, but no zearalenone in the hay. Occurrence of mycotoxins such as T-2 and HT-2 in the moldy hay coincided with, and may have been responsible for the impaired semen quality in AI bulls. This case report will draw the attention to the possible hazards when feeding moldy hay.

Polyamine depletion in human melanoma cells leads to G1 arrest associated with induction of p21WAF1/CIP1/SDI1, changes in the expression of p21-regulated genes, and a senescence-like phenotype.

The cell cycle regulatory events that interface with polyamine requirements for cell growth have not yet been clearly identified. Here we use specific inhibitors of polyamine biosynthetic enzymes to investigate the effect of polyamine pool depletion on cell cycle regulation. Treatment of MALME-3M cells with either the ornithine decarboxylase inhibitor alpha-difluoromethylornithine or the S-adenosylmethionine decarboxylase inhibitor MDL-73811 lowered specific polyamine pools and slowed cell growth but did not induce cell cycle arrest. By contrast, treatment with the combination of inhibitors halted cell growth and caused a distinct G1 arrest. The latter was associated with marked reduction of all three polyamine pools, a strong increase in p21(WAF1/CIP1/SDI1) (p21), and hypophosphorylation of retinoblastoma protein. All effects were fully prevented by exogenous polyamines. p21 induction preceded p53 stabilization in MALME-3M cells and also occurred in a polyamine-depleted, p53-nonfunctional melanoma cell line, indicating that p21 is induced at least in part through p53-independent mechanisms. Conditional overexpression of p21 in a fibrosarcoma cell line was shown previously to inhibit the expression of multiple proliferation-associated genes and to induce the expression of genes associated with various aspects of cell senescence and organism aging. Polyamine depletion in MALME-3M cells was associated with inhibition of seven of seven tested p21-inhibited genes and with induction of 13 of 14 tested p21-induced genes. p21 expression is also known to induce a senescence-like phenotype, and phenotypic features of senescence were observed in polyamine-depleted MALME-3M cells. Cells increased in size, appeared more granular, and expressed senescence-associated beta-galactosidase. Cells released from the polyamine inhibition lost the ability to form colonies, failed to replicate their DNA, and approximately 25% became bi- or multinucleated. These events parallel the outcome of prolonged p21 induction in fibrosarcoma cells. The results of this study indicate that polyamine pool depletion achieved by specific biosynthetic enzyme inhibitors causes p21-mediated G1 cell cycle arrest followed by p21-mediated changes in gene expression, development of a senescence-like phenotype, and loss of cellular proliferative capacity.

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.

Calcification of the intervertebral discs and curvature of the radius and ulna: a radiographic survey of Finnish miniature dachshunds.

The vertebral column of 124 randomly selected miniature dachshunds, representing 4.5% of the population registered by the Finnish Kennel Club during the years 1988 to 1996, were radiographed. The front legs were also radiographed in order to evaluate the curvature of the radius and ulna. Calcified discs were found in 75.9% of the longhaired miniature dachshunds and in 86.7% of the wirehaired ones. The occurrence of signs associated with IDD was 16.5% in longhaired and 15.6% in wirehaired miniature dachshunds. The occurrence of signs of IDD in dogs with calcified discs was 20.0% and 17.9% in longhaired and wirehaired miniature dachshunds, respectively. In dogs without calcifications only one dog showed signs of IDD. The curvature of the radius and the ulna did not differ between the dogs with signs of IDD and the healthy ones, or between the dogs with and without intervertebral calcifications. Our results indicate that radiographic eradication based on the presence of intervertebral calcifications is not suitable for breeding purposes for the Finnish miniature dachshund population because the percentage of dogs without calcifications is small.

The organization of replicon clusters is not affected by polyamine depletion.

Earlier investigations have shown that polyamine depletion affects DNA replication negatively. DNA is synthesized in replicons which are gathered in replicon clusters. DNA replication is initiated simultaneously in every replicon of a replicon cluster. By pulse labeling cells with the thymidine analog bromodeoxyuridine and then detecting bromodeoxyuridine in situ with immunofluorescence, replicon clusters can be studied. We have used this method to investigate the effects of 2-difluoromethylornithine (DFMO)- and 4-amidinoindan-1-one 2'-amidinohydrazone (CGP 48664)-mediated polyamine depletion on the organization of replicon clusters. The cells were studied by fluorescence microscopy and confocal laser scanning microscopy. Our studies give at hand that neither the number nor the distribution of replicon clusters were affected even after 4 days of treatment with 5 mM DFMO or 20 microM CGP 48664, indicating that polyamine depletion did not affect the organization of replicon clusters. However, the fluorescence intensity of the replicon clusters was much lower in inhibitor-treated cells. The results indicate that the impaired DNA replication observed in polyamine-depleted cells is not due to an effect on the initiation step of DNA replication, but rather on the elongation process. To confirm that it is possible to observe changes in the organization of replicon clusters using bromodeoxyuridine, we treated the cells with various drugs that affect DNA replication. Aphidicolin, which inhibits DNA elongation, gave results similar to those of DFMO and CGP 48664.

Treatment of cells with the polyamine analog N, N11-diethylnorspermine retards S phase progression within one cell cycle.

When Chinese hamster ovary cells were seeded in the presence of the spermine analog N1,N11-diethylnorspermine (DENSPM), cell proliferation ceased; this was clearly apparent by cell counting 2 days after seeding the cells. However, 1 day after seeding there was a slight difference in cell number between control and DENSPM-treated cultures. To investigate the reason for this easily surpassed slight difference, we used a sensitive bromodeoxyuridine/flow cytometry method. Cell cycle kinetics were studied during the first cell cycle after seeding cells in the absence or presence of DENSPM. Our results show that DENSPM treatment did not affect the progression of the cells through G1 or the first G1/S transition that took place after seeding the cells. The first cell cycle effect was a delay in S phase as shown by an increase in the DNA synthesis time. The following G2/M transition was not affected by DENSPM treatment. DENSPM treatment inhibited the transient increases in putrescine, spermidine, and spermine pools that took place within 24 h after seeding. Thus, in conclusion, the first cell cycle phase affected by the inhibition of polyamine biosynthesis caused by DENSPM was the S phase. Prolongation of the other cell cycle phases occurred at later time points, and the G1 phase was affected before the G2/M phase.

Half-lives of ornithine decarboxylase and S-adenosylmethionine decarboxylase activities during the cell cycle of Chinese hamster ovary cells.

Cells in mitosis were seeded immediately after being harvested by the mitotic shake off technique from a culture of exponentially growing Chinese hamster ovary cells. At 2, 5, 7, 10, and 12 h after seeding, cycloheximide was added. Cells were sampled at various times after cycloheximide addition and the ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC) activities were determined. Flow cytometric analysis showed that cells sampled at 2, 5, 7, 10, and 12 h after seeding were found in mid G(1), at the G(1)/S transition, in mid S phase, at the S/G(2) transition, and in late G(2), respectively. The half-lives of ODC and AdoMetDC activities varied during the cell cycle. The half-life of ODC activity showed a biphasic pattern with increases in connection to the G(1)/S and S/G(2) transitions while the half-life of AdoMetDC activity increased only at the G(1)/S transition.

Topoisomerase II is nonfunctional in polyamine-depleted cells.

The polyamines-putrescine, spermidine, and spermine-are essential for normal cell proliferation. Polyamine depletion affects DNA structure and synthesis. Topoisomerase II (topo II) is also necessary for normal cell proliferation, and it has been shown in vitro that polyamines may affect topo II activity. In order to investigate the effect of polyamine depletion on topo II activity, we treated Chinese hamster ovary cells with either alpha-difluoromethylornithine (DFMO) or 4-amidinoindan-1-one-2'-amidinohydrazone (CGP 48664), which are polyamine biosynthesis inhibitors. Treatment with the topo II inhibitor etoposide results in DNA strand breaks only if there is active topo II in the cells. By quantitating DNA strand breaks after etoposide treatment using single cell gel electrophoresis, we were able to estimate intracellular topo II activity. We also quantitated topo II activity in crude nuclear extracts from control and polyamine biosynthesis inhibitor-treated cells. Using single cell gel electrophoresis, we noted a clear decrease in the function of topo II in polyamine biosynthesis inhibitor-treated cells, as compared with untreated control cells. However, the topo II activity in crude nuclear extracts did not differ significantly in control versus polyamine biosynthesis inhibitor-treated cells. Taken together, these results indicate that although the function of topo II in polyamine-depleted cells was impaired, topo II remained functional in an in vitro assay. Using the single cell gel electrophoresis assay, we also found that spermine depletion itself caused DNA strand breaks.