Traumatic cervical spinal cord injury: a prospective clinical study of laryngeal penetration and aspiration.

STUDY DESIGN: Prospective cohort study. OBJECTIVES: Dysphagia is a relatively common secondary complication in patients with traumatic cervical spinal cord injuries (TCSCI). The purpose of this study was to determine the incidence of aspiration and penetration in patients with acute TCSCI. SETTING: Tampere University Hospital, Tampere, Finland. METHODS: A total of 46 patients with TCSCI were evaluated with a videofluoroscopic swallowing study (VFSS). Rosenbek's penetration-aspiration scale (PAS) was used to classify the degree of penetration or aspiration. The medical records of each patient were systematically reviewed. RESULTS: Of the 46 patients, 85% were male. The mean age at the time of the injury was 62.1 years. Most patients had an incomplete injury (78%), and most of them due to a fall (78%). In the VFSS 19 (41%) patients penetrated and 15 (33%) aspirated. Only 12 (26%) of the patients had a PAS score of 1 indicating that swallowed material did not enter the airway. Of the patients who aspirated, 73% had silent aspiration. CONCLUSION: The incidence of penetration or aspiration according to VFSS is high in this cohort of patients with TCSCI. Therefore, the swallowing function of patients with acute TCSCI should be routinely evaluated before initiating oral feeding. VFSS is highly recommended, particularly to rule out the possibility of silent aspiration and to achieve information on safe nutrition consistency.

Centralized spinal cord injury care in Finland: unveiling the hidden incidence of traumatic injuries.

STUDY DESIGN: Population-based prospective study. OBJECTIVES: To determine the incidence and evaluate the characteristics of newly injured patients with traumatic spinal cord injury (TSCI) admitted to two of the three national spinal cord injury (SCI) centers during the first year after the centralization of SCI care in Finland. SETTING: Oulu and Tampere University Hospital SCI centers, Finland. METHODS: The designated rehabilitation teams evaluated all of the patients with a new SCI and persisting neurological symptoms. The data were recorded according to the International Spinal Cord Injury Core Data Set. RESULTS: In a 1-year period, 77 new patients with TSCI were admitted to the study centers serving a population of 3 065 946. In the whole catchment area, the mean annual incidence of TSCI was 25.1 per million, and in the hospital districts of the SCI centers, the incidence was even higher, at 38.1 per million. The mean age of the patients was 58.7 years. Falls were the leading cause of injury (64.9%), and the injury resulted in tetraplegia in 70.1% of the cases. Alcohol use was a contributing factor in 39% of the cases in the entire sample and in 58.6% of cases among patients aged younger than 60 years. CONCLUSION: The incidence rates of TSCI were markedly higher than expected, demonstrating the previously hidden morbidity. The epidemiological features of TSCI appeared to follow the trends in developed countries, highlighting the increasing incidence of cervical lesions due to falling among the elderly. The results need to be confirmed in an extended follow-up.

Clinical correlates of cerebral diffusion tensor imaging findings in chronic traumatic spinal cord injury.

STUDY DESIGN: Prospective clinical case-control study. OBJECTIVES: The aim of this study was to use diffusion tensor imaging (DTI) to assess the state of cerebral white matter tracts after spinal cord injury (SCI). The DTI metrics were evaluated in relation to neurological deficits and to the size and level of the spinal cord lesions. SETTING: Tampere University Hospital, Tampere, Finland. METHODS: Thirty-four patients (n=34) with clinically complete and incomplete SCI were evaluated using the International Standards for Neurological Classification of Spinal Cord Injury (ISNCSCI). DTI metrics (fractional anisotropy (FA) and apparent diffusion coefficient (ADC)) were calculated for multiple levels along the course of the corticospinal tract. The state of the spinal cord after injury was assessed using conventional magnetic resonance imaging (MRI). DTI parameters were compared with 40 orthopedically injured control subjects. RESULTS: Statistically significant differences in the DTI values between patients and controls were detected in the posterior area of the centrum semiovale. In this area, the FA values were lower in the patients compared with controls (P=0.008). For patients with clinically complete injury, the difference was even more significant (P=0.0005). Motor and sensory scores of the ISNCSCI correlated positively with FA and negatively with ADC values of the centrum semiovale. A moderate association was observed between the macroscopic changes in the spinal cord and the DTI abnormalities in the centrum semiovale. CONCLUSION: In patients with chronic SCI, DTI changes can be observed in the cerebral white matter. These alterations are associated with the clinical state of the patients.

Mid-term results for three contemporary total knee replacement designs - a comparative study of 104 patients with primary osteoarthritis.

AIMS: The purpose of this study was to compare retrospectively the mid-term clinical and radiological results of three contemporary knee designs in cohorts operated on in the same hospital during the same time period. MATERIALS AND METHODS: We evaluated mid-term clinical and radiographic outcome of three contemporary total knee designs (the AGC V2, the Duracon and the Nexgen) in 104 consecutive patients (129 knees) operate on for primary knee osteoarthritis at our hospital. The mean indexed age at the time of the operation was 69.2 years (range, 49.3 to 81.1 years). The mean follow-up time was 6.0 years (range, 0.2 to 7.9). All patients were followed for at least three years or until the first revision. In the survival analyses, the end point was defined as, revision for any reason. RESULTS: The Kaplan-Meier survival analysis showed a 98% (95% CI 94-100) survival rate for the NexGen, a 98% (95% CI 93-100) for the AGC and a 90% (95% CI 81-99) for the Duracon design at six years. Both the mean KSS for pain, KSS for function and the mean clinical knee score improved significantly in all three groups. There was no difference between the three designs in mid-term survivorship. CONCLUSIONS: Most of the revisions could be directly linked to perioperative surgical errors. In conclusion, the most recently introduced knee replacements of the present study (Duracon and Nexgen) did not show any clinically significant benefit over the older design (AGC) in the mid-term.

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.

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 > or = 20 mares; in addition, stallions with foaling rates < 60 or > 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.

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.

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.

Biophysical and 1H magnetic resonance spectroscopy characteristics of fractionated stallion ejaculates.

The composition of seminal plasma must be determined to assess the possible roles of sex gland secretions in survival of stallion spermatozoa. In the present study, an automated semen collection device and 1H magnetic resonance spectroscopy were used to analyse and compare the composition of seminal plasma from fractionated and nonfractionated stallion ejaculates. The contribution of each semen component to the ejaculate (sequence of production of component and concentration) was evaluated and its relationship to biophysical parameters was determined. 1H magnetic resonance spectroscopy was used to quantify molecules defined as markers of sex gland secretions: carnitine, glycerophosphorylcholine and choline for the epididymides; N-acetyl function of glycoproteins and spermine for the ampullae; acetic acid for the bulbourethral glands; and citric acid for seminal vesicles. The results from 32 ejaculates (four ejaculates from each of four stallions by two collection methods) demonstrated the reliability of the 1H magnetic resonance spectroscopy quantitation, the sequence of sex gland secretion contributions to the ejaculate (bulbourethral glands, epididymides, ampullae and seminal vesicles) and the concomitant appearance of the sperm-rich fraction with secretions from the epididymides and ampullae.

Freezing of stallion semen with addition of glycine betaine.

The effect of addition of glycine betaine to a lactose-EDTA freezing medium on the post-thaw motility of stallion semen was determined. The first three semen-rich fractions of nine stallions were collected with an open-end Krakow artificial vagina on consecutive weekdays. Semen was frozen using the Hannover method with freezing media containing glycine betaine in various concentrations from 0 to 5%. After thawing, sperm motility was analysed both by a light microscope and by a Hamilton-Thorn Motility Analyser. Total and progressive post-thaw motilities of semen containing 0.25-3% glycine betaine did not differ significantly from the total and progressive post-thaw motilities of semen frozen without glycine betaine. The total and progressive post-thaw motilities of semen containing 4 or 5% glycine betaine were significantly lower (P < 0.001) than those of semen without glycine betaine. In conclusion, glycine betaine did not show any beneficial effect on the post-thaw motility of stallion semen when semen was frozen using the Hannover method.

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.

Some effects of multiple administration of detomidine during the last trimester of equine pregnancy.

Detomidine was given to 11 pregnant mares at 3 week intervals during the last trimester of pregnancy. Maternal and fetal electrocardiographs were recorded and fetal activity studied by transabdominal ultrasonography, before and 2 h (2, 5, 10, 20, 30, 60, 90 and 120 min) after injection. After parturition, the foals were examined and weighed. Maternal and fetal heart rate showed an initial decline after detomidine administration. Maternal heart rate in the treatment group were lower already 2 min after injection, but a reduction in fetal heart was first seen 5 min after detomidine administration. Mean fetal heart rate at 2 min after detomidine injection was 109, 104, 95 and 90 beats/min, whereas at 5 min it was 80, 76, 72 and 66 beats/min in the 2nd, 3rd, 4th and 5th examination session, respectively. The heart rates did not revert to the control values during follow-up. Decline and recovery patterns were quite similar during all examination sessions. The mares exhibited conductive disturbances 2 min after detomidine administration, but fetal heart rhythm remained regular. Fetal activity was decreased at 5 min but had reverted to control values about 90 min after detomidine administration. Administration of detomidine (0.015 mg/kg) to healthy pregnant mares at 3 week intervals during the last trimester had no measurable detrimental effects on the outcome of pregnancy.

Effect of 19-norandrostenololylaurate on semen characteristics of colts.

The long-term effects of the anabolic steroid 19-norandrostenololylaurate on semen characteristics of Finnhorse colts were studied in 3 experiments. Semen was collected initially at 24 months of age and then twice a year. In experiment I, 500 mg or 100 mg of steroid per animal was given every 3rd week from 12 or 16 months to 24 months of age. In colts treated with 500 mg of anabolic steroid every 3rd week, azoospermia was observed in 3 out of 5 colts in the first semen collections, immediately after the end of treatment. The other 2 colts had low sperm numbers and a high percentage of proximal droplets in relation to control animals. The 100 mg group was less affected by steroid treatment than the 500 mg group. The seminal changes were observed to be reversible. All of the colts had spermatozoa in their ejaculates 4 months after the end of treatment. Two years after cessation of treatment, sperm numbers in treated animals exceeded those in the control group. In experiment II, colts were treated from 7 months to 12 months of age with a dose of 1 mg/kg every 3rd week or 0.3 mg/kg every week. The first semen collections took place 12 months after the last treatment. The animals treated with 0.3 mg/kg every week were less affected than those treated with 1 mg/kg every 3rd week. In experiment III, animals were treated from 3 months to 8 months of age, and the interval from the last treatment to the first semen collection was 16 months. There were no significant differences between the groups in any of the semen parameters examined. In the last semen collection, the control animals had numerically higher spermatozoal concentration and progressive motility and less dead sperm than the treated animals. It was concluded that the adverse effects of steroid treatment on semen characteristics were reversible, at least in the groups treated at age 7-25 months.

Effect of 19-norandrostenololylaurate on testicular growth in colts.

The effect of anabolic steroid on testicular growth was investigated in 3 experiments. In experiment I, 500 mg of the anabolic steroid was given to 4 colts and 100 mg to another 4 colts, every 3rd week, starting at age 16 months and ending at age 24 months. Six colts served as controls. Both treatments decreased total scrotal width (TSW) within 6 weeks. Seasonal testicular growth during spring partly overcame the effect of steroid treatment. Cessation of anabolic steroid treatment was followed by testicular growth at the same time as TSW in untreated colts was decreasing by virtue of the effect of the season. Four months after the last injection, TSW was smaller in the treated animals than in the untreated animals, but the difference was not statistically significant. In experiment II, steroid was given at a dose of 1 mg/kg every 3rd week to 4 colts and 0.3 mg/kg every week to 4 colts. Six colts served as controls. The animals were 7 months old at the start of treatment and 12 months old at the end of treatment. Treatments decreased testicular widths (TW), within 6-9 weeks. In this experiment, also, cessation of anabolic steroid treatment was followed by testicular growth. Twelve months after the last treatment, TW was smaller in treated than in untreated animals but the difference was not statistically significant. In experiment III, foals were used which were 3 months old at the start and 8 months old at the end of treatment. The steroid was given at a dose of 1 mg/kg every 3rd week to 3 foals. Three foals served as controls. Treatment decreased TW within 6 weeks. Cessation of treatment was followed by a slow testicular growth. Growth similar to that in control animals started after a delay of 4-5 months. TW in treated animals nearly reached TW in controls within 12 months of cessation of treatment.

Intrauterine fluid accumulation in oestrous mares.

Intrauterine fluid (IUF) was collected using a tampon from mid-oestrous mares (n = 57) with and without ultrasonically detectable accumulations of free intraluminal fluid. Bacteria were cultured and neutrophils counted from all samples (n = 57). Total protein concentration, trypsin-inhibitor capacity (TIC), and plasmin, beta-glucuronidase (B-Gase) and N-acetyl-beta-D-glucosaminidase (NAGase) activities were determined in 27 IUF samples. The motility of spermatozoa in the presence of IUF, IUF extended with Kenney's medium (1:1) and Kenney's medium alone was analysed in 9 samples using a Hamilton-Thorn motility analyser. Thirty-five mares were inseminated immediately after collection of IUF, and every second day until ovulation. Embryos were recovered nonsurgically 6 days after ovulation. After embryo transfer, fluid accumulations were recorded during oestrus and an endometrial biopsy specimen taken (n = 53). In the beginning of oestrus, fluid accumulations were detected in 39% (22/57) of mares, while on the day when IUF was collected, fluid accumulations were observed in 26% (15/57) of mares. The fluid was anechogenic, and in 80% of the mares located in the uterine body. None of the mares exhibited cytological or bacteriological evidence of acute endometritis. Total protein concentrations, TIC and B-Gase activities in IUF were statistically significantly lower in mares with fluid accumulations (n = 14) than in mares without fluid accumulations (n = 13) (p < 0.01). The addition of undiluted IUF to extended semen significantly reduced total and progressive motilities, path velocities and percentages of rapid spermatozoa (p < 0.05) in vitro. On endometrial biopsy, fibrosis was found to be more prominent (p = 0.025) in mares with fluid accumulations (n = 9) than in mares without (n = 44). It was concluded that anechogenic fluid accumulations during oestrus were associated with compositional changes in IUF. Although IUF had negative effects on spermatozoal motility in vitro, the presence of fluid accumulations at the time of insemination did not affect embryo recovery rates.

Effect of 19-norandrostenololylaurate on serum testosterone concentration, libido, and closure of distal radial growth plate in colts.

The long-term effect of anabolic steroid was investigated in 3 experiments. In experiment I, 500 mg of 19-norandrostenololylaurate was given to 5 colts and a dose of 100 mg to another 5 colts every 3rd week. Six colts served as untreated controls. The animals were 12-16 months old at the start, and 24 months at the end of treatment. In experiment II, a dose of 1 mg/kg was given every 3rd week to 4 colts and 0.3 mg/kg every week to another 4 colts. Six colts served as controls. The colts were treated from 7 months to 12 months of age. In experiment III, 1 mg/kg of steroid was given every 3rd week to 3 foals between 3 and 8 months of age. Three foals were used as controls. Libido and hCG-induced serum testosterone concentrations were studied after the cessation of treatments, up to 3 or 4 years of age. Closure of the right distal radial growth plate was determined between 21 and 36 months of age. Treated colts had lower testosterone levels 4.5 months after cessation of treatment in experiment I (p < 0.001) and experiment II (p < 0.05) when compared with the corresponding control groups. Two years after treatments in experiment I, hCG-induced testosterone levels were higher in treated colts than in untreated controls, but the difference was not statistically significant. The treatments had no effect on libido. The anabolic steroid treatment did not cause premature closure of epiphyseal growth plates in any of the experiments, but closure appeared to be delayed. It was concluded that anabolic steroids have long term effects on reproduction. Their influence on serum testosterone can last for years after cessation of treatment, and they can delay the closure of growth plates which can cause increased susceptibility to cartilage injury during exercise.

Effect of anti-freeze protein (AFP) on the cooling and freezing of equine embryos as measured by DAPI-staining.

Equine embryos recovered on Day 6 after ovulation were cooled to +4 degrees C, or frozen with AFP alone or together with glycerol. Twenty embryos (140-200 microm in diameter) were randomly assigned to 6 treatment groups. In the first 3 groups, the embryos were cooled from room temperature to +4 degrees C at a rate of 3 degrees C/min and warmed again at a rate of 32 degrees C/min in a programmable freezer. In the second 3 groups, the embryos were frozen using a standard protocol, stored in liquid nitrogen for 5-7 days and then thawed in a 37 degrees C waterbath. After cooling/warming or freezing/thawing all the embryos were stained with DAPI. The percentage of dead cell area was significantly lower in the cooling groups than in the freezing groups and no significant differences were apparent between the cryoprotectants used in the study.

The effect of sucrose in the thawing solution on the morphology and mobility of frozen equine embryos.

Seventy-five embryos were collected 6 days after ovulation. Sixty embryos were frozen in straws using glycerol as the cryoprotectant in an automatic freezer. In Experiment 1 the freezing and thawing media were supplemented with 1.3 g/l PVP; in Experiment 2 the supplement was 5% FCS. The embryos were thawed for 30 s at +37 degrees C in a waterbath. In Experiment 1 glycerol was removed from 10 embryos in 6 steps. In 10 other embryos, glycerol and sucrose were both removed from the medium in 6 steps. After glycerol and sucrose removal, the embryos were stained with 4',6'-diamidino-2-phenylindole (DAPI) to count the percentage of dead cells. Fluorescent rate (FR) was defined as a ratio of fluorescent area versus total area. Mean (+/- s.d.) FR in this experiment was significantly lower (P<0.01) in embryos thawed with sucrose (0.28 +/- 0.13) than in embryos thawed with glycerol alone (0.53 +/- 0.25). In Experiment 2, 40 embryos were frozen and glycerol, with or without sucrose, was removed after thawing as for Experiment 1. Ten embryos in both groups were stained with DAPI. All the frozen-thawed embryos were transferred nonsurgically to recipient mares. Fourteen fresh embryos were transferred as controls, 7 of which were stained with DAPI before transfer. There was no difference in pregnancy rates between DAPI-stained versus nonstained embryos, indicating that the staining process had no negative effects on embryonic survival. Insufficient embryos were transferred to be able to demonstrate any difference in pregnancy rates between embryos thawed with or without sucrose in the medium.

Recovery rate and quality of embryos from mares inseminated after ovulation.

The aim of this study was to evaluate the quality of embryos and their recovery rate from mares inseminated at different intervals after ovulation. Finnhorse and warmblood mares were inseminated with fresh semen 8 to 16 h, 16 to 24 h, or 24 to 32 h after ovulation. Control mares were inseminated before ovulation. Sixty-seven embryo flushings were performed between Days 7 and 9 after ovulation/insemination. Thirteen mares were not flushed, but their uteri were scanned for pregnancy on Days 14 to 16. Embryo recovery rates decreased as time from ovulation to insemination increased, although embryo quality remained normal as evaluated by morphological criteria and mitotic index. However, postovulatory insemination in this trial appeared to delay embryo development, since the embryos recovered from mares inseminated after ovulation were appreciably smaller and at an earlier stage of development than control embryos recovered from mares inseminated prior to ovulation. Part of this delay in embryo development in the postovulation group could be due to the time needed for sperm capacitation. In addition, as the time from ovulation to insemination increased, embryo development might have been further delayed by defects in the aging oocyte.

Effect of timing of frozen semen insemination on pregnancy rate in mares.

Thirty-four mares were inseminated with frozen semen from one stallion during 2 oestrous cycles, every 48 h until ovulation took place and within 12 h after ovulation. Semen was frozen using the Colorado method. The insemination dose was from 200 to 400 x 10(6) progressively motile spermatozoa. Ovaries were examined every 12 h to determine time of ovulation. Examination for pregnancy was carried out using ultrasonography, 15 days after ovulation. Thirty-five per cent of mares inseminated < 24 h and 23% of mares inseminated between 24-48 h before ovulation were pregnant (p = 0.388). The pregnancy rate in all mares inseminated before ovulation was 30%. In the mares inseminated within 12 h of ovulation, it was 18% (p = 0.253). Younger mares (aged 4-10 yr) had a higher pregnancy rate (59%) than older mares (aged 11-15 yr) (23%), but the difference was not statistically significant (p = 0.057).