Validation of the FACSCount AF System for Determination of Sperm Concentration in Boar Semen

A flow cytometric method has been developed for rapid determination of sperm concentration in semen from various mammalian species. * * Patent Pending, Int. Publication Number WO/00/54026.
All cells containing DNA are stained with SYBR‐14 or propidium iodide (PI) and sperm concentration is determined in relation to an internal standard of fluorescent microspheres (beads). Satisfactory staining can be achieved within 2–3 min and the following flow cytometric analysis on the FACSCount AF System rapidly provides the user with a precise and accurate assessment of the sperm concentration. In this study, the FACSCount AF System and Sperm Counting Reagent (BD Biosciences) was compared with microscopic counting using a Bürker–Türk haemocytometer. In addition, sperm concentration was determined using the Corning 254 spectrophotometer which is used routinely by Danish artificial insemination stations for boars. The results show that the agreement between flow cytometry and microscopic counting is very high. The slope for the regression line was 1.12 (SE = 0.03) with an estimated intercept with the Y‐axis of 22 × 10⁶sperm/ml (SE = 10 × 10⁶ sperm/ml) and an estimated error of the model of 10 × 10⁶ sperm/ml. For the spectrophotometer, the slope of the regression line was 1.09 (SE = 0.07) with an estimated intercept of 137 × 10⁶ sperm/ml (SE = 25 × 10⁶ sperm/ml). The average error made by the spectrophotometer was 55 × 10⁶ sperm/ml. In addition, the results obtained using flow cytometry was highly repeatable (CV = 2.7%) in comparison with the spectrophotometric method (CV = 6.3%). These results indicate that the FACSCount AF System is a valuable tool for precise and accurate assessment of sperm concentration in boar semen and that use of this system may lead to production of more uniform insemination doses containing a specific number of sperm per dose.     

Management Strategies Aiming to Improve Horse Welfare Reduce Embryonic Death Rates in Mares

The objective of this retrospective study was to evaluate the effect of management strategies aiming to improve animal well‐being on pregnancy and embryonic death (ED) rates. Breeding records of a cohort of 1206 Thoroughbred mares brought to a stallion station facility, to be bred with the stallions housed there, were evaluated during ten breeding seasons. Mares were blocked according to management strategies in two groups: Stress and Relax. Strategies used to improve animal well‐being (Relax group) were as follows: stopping the teasing routine, reducing or eliminating stall confinement, reducing the number of mares per group and maintaining herd stability during the breeding season. In barren mares, the pregnancy rate was higher in the Relax group (91.8%) when compared to the observed in Stress group (84.7%). However, no difference in pregnancy rates were observed (Stress = 85.2% vs. Relax = 86.2) in foaling mares. ED rate was higher in barren and foaling mares of the Stress group mares (25.5% and 26.8%, respectively) compared with the Relax group (16.1% and 14.7%, respectively). No significant differences were observed on foal heat pregnancy rate between groups; yet, the embryo loss on foal heat was significant reduced in Relax mares (Relax = 8.7% vs Stress = 24.5%). In conclusion, management strategies aimed to reduce social stress can reduce early pregnancy losses and the average cycles per pregnancy, improving reproductive performance in mares.     

The effect of autonomic manipulation on heart rate variability in dogs

Beat‐to‐beat variation of heart rate is reflective of autonomic balance and has been used to assess pain and stress in human beings. The purpose of this investigation was to pharmacologically manipulate the autonomic nervous system and to determine the effect of these manipulations on heart rate variability (HRV) in dogs. Four adult male hound dogs (27 ± 1 kg) were used in the investigation. Each dog was given five treatments: Parasympathetic blockade (glycopyrrolate; 0.01 mg kg^–1 IV and 0.01 mg kg^–1 IM), parasympathetic stimulation (phenylephrine; 0.005 mg kg^–1 IV + 0.05 mg kg^–1hour^–1), sympathetic blockade (propranolol; 0.11 mg kg^–1 IV), sympathetic stimulation propranolol; 0.01 μg kg^–1 minute^–1), and saline control. At least 48 hours were allowed between treatments. ECG recordings were obtained using an ambulatory ECG monitor. A 5‐minutes period of continuous recording obtained ~30 minutes after initiation of drug administration was used for data analysis. Changes in HRV were evaluated by time and frequency‐domain analysis. The standard deviation of normal R‐R intervals (SDNN), as well as the standard deviation of successive differences in RR intervals (SDSD) were assessed for each treatment. Low frequency (LFP; 0.05–0.15 Hz), high frequency (HFP; 0.15–0.35 Hz), and total (TP; 0.017–0.5 Hz) spectral power were also determined. The LFP:HFP ratio was also evaluated. A two‐way anova with a Tukey's test was used to detect differences (p < 0.05). Administration of glycopyrrolate or isoproterenol increased HR and decreased SDNN and SDSD below control levels. Phenylephrine or propranolol administration were without effect. LFP was diminished by glycopyrrolate and isoproterenol, but was unaffected by phenylephrine and propranolol. HFP, TP, and LFP:HFP were unaffected by treatment. Both branches of the autonomic nervous system influence SDNN and LFP. SDSD, in contrast, is altered primarily by parasympathetic activity. Thus, it appears that parasympathetic activity modulates HRV in the resting dog, as either withdrawal of parasympathetic influence or accentuated sympathetic activity led to significant changes in these measures of HRV. Conversely, augmentation of parasympathetic activity or withdrawal of sympathetic tone minimally affected HRV.     

Unit activity: motiviation-dependent responses from midbrain neurons

Single neurons in the midbrain tegmentum of rats showed clear discriminatory responses to three tones announcing either food or water or no reinforcement. Subjects were required to press a single lever and remain motionless for 2 seconds during which time unit activity from several brain sites was recorded. One of the tones, randomly designated, was sounded halfway through this period. Manipulation of drive states revealed that the highest activity was associated with tones announcing the reinforcement for which the animal was most strongly motivated. Thus, from a hungry animal sustained or increased firing rates were elicited by a " food" tone and differentially lowered rates by the other tones.