Synchronizing Cell Cycle of Goat Fibroblasts by Serum Starvation Causes Apoptosis

Cell cycle stage and synchronization of donor cells are important factors influencing the success of somatic cell nuclear transfer. This study examined whether serum starvation has any effect on specific cell death. We also studied the effects of serum starvation, culture to confluence, and full confluency (confluent + 72 h) on cell cycle characteristics and apoptosis of goat dermal fibroblast cells. The cells were obtained from the ear of a 1.5‐year‐old female goat. The following experimental groups were analysed for fibroblast cells: (i) normally growing, (ii) confluent, (iii) full confluency, (iv) cells starved for 48 h and (v) cells starved for 72 h. Analysis of cell cycle distribution by flow cytometry showed that 4.56 and 51.88% of normal cycling cells were at the G0 and G1 phases respectively. In the confluent group, 80% of the cells were arrested in the G0/G1 phase. Serum starvation for 48 and 72 h arrested 84.78% and 90.1% cells at the G0/G1 phase respectively which showed a significant difference when compared with the control group (p < 0.05). Double staining by PI and FITC distinguishes G0 phase from G1 phase. In the full confluency group, 91.53% of cells were at G0/G1 stage, but in contrast to the serum starved group, this high percentage of G0/G1 cells was mainly associated with G1 cells. Under normal culture conditions, 6.39% of cells underwent early apoptosis. In the confluent group 8.93% of cells showed early apoptosis. Serum starvation for 48 and 72 h caused early apoptosis in 8.91 and 39.83% of the cells respectively. Full confluency treatment did not increase the number of apoptotic cells significantly (8.67%). After 72 h, serum starvation significantly increased early apoptosis (p < 0.05). In conclusion, the use of full confluency is suitable for cell cycle synchronization because it arrests cells at the G0/G1 phase and also induces less apoptosis in comparison with the serum starvation group.     

Evaluation of a Neck Mounted 2‐Hourly Activity Meter System for Detecting Cows About to Ovulate in Two Paddock‐Based Australian Dairy Herds

Two studies were conducted to assess the performance of a commercially available neck‐mounted activity meter to detect cows about to ovulate in two paddock‐based Holstein‐Friesian dairy herds. The activity monitoring system recorded cow activity count in 2‐hourly periods. Study I investigated the ability of the system to detect cow ovulatory periods in dairy herds managed in two different Australian environments and breeding systems using five activity alert algorithms. Herd 1 consisted of approximately 130 milking cows calving year‐round in a sub‐tropical environment and kept in a single dry lot paddock. Herd 2 consisted of approximately 400 milking cows calving seasonally in a temperate climate and fed pasture by rotation through multiple grazing paddocks. Ovulatory periods and non‐ovulatory days were identified using milk progesterone monitoring alone or in combination with ovarian ultrasonography; using these ‘gold standards’ 141 and 135 ovulatory periods were identified in 64 and 135 cows in Herds 1 and 2 respectively. Sensitivity of the activity monitoring system for detecting cow ovulatory periods ranged from 79.4% to 94.1%, specificity from 90.0% to 98.2% and positive predictive value from 35.8% to 75.8%. Study II investigated the ability of the activity meter system to predict the timing of ovulations in paddock‐based pasture‐fed dairy cattle (Herd 2). The time of ovulation was estimated by repeat trans‐rectal ovarian ultrasonography at approximately 0, 12, 24 and 36 h after artificial insemination (AI). The mean times (±SD) from onset and end of increased activity to ovulation were 33.4 ± 12.4 and 17.3 ± 12.8 h respectively (n = 94). Fifty per cent of cows (n = 47) ovulated within the 8‐h period between 30 to 38 hs after the onset of increased activity, 76.6% (n = 72) within the 16 h between 24 to 40 h, 85.1% (n = 80) within the 24 h between 18 and 42 h and 90.4% (n = 85) within the 32 h from 19 to 51 h after the onset of increased activity. Results from these studies show that in paddock‐based dairy cows in two diverse management systems, this neck‐mounted activity meter system detects high proportions of cows that are about to ovulate and provides a useful indication of when ovulation is likely to occur. However, the specificities and positive predictive values using the algorithms assessed may be lower than desirable.     

Confirmation of epidural needle placement using nerve stimulation in dogs

Caudal epidural anesthesia is useful when anesthesia of the lumbar and sacral dermatomes is needed. Its success relies on the proper placement of the needle in the epidural space. However, accurate positioning of the needle can be difficult in certain patients (i.e.obesity). The purpose of this preliminary study was to document the use of nerve stimulation as a means of confirming accurate needle positioning in the epidural space prior to drug administration. Twenty large breed dogs undergoing hindlimb or perineal surgery were enrolled. Following induction of general anesthesia, patients were prepared for routine epidural drug administration. A 17 ga, 3.5” shielded Tuohy needle was used and was connected to a peripheral nerve stimulator set to deliver a current at 1 Hz, with a pulse width of 0.2 m sec. Initial current was set at 1.2 mA as the needle was advanced into position. Confirmation of epidural needle placement was confirmed when twitches were observed in the hindlimbs and/or tail. Current setting was then decreased incrementally by 0.2 mA until no further twitches were observed. Success of epidural drug placement was confirmed subjectively by motor blockade to the blocked dermatomes and clinical signs of balanced anesthesia (lack of sympathetic response to surgical stimulation while maintained at light plane of anesthesia). Lowest mean (range) current to elicit hindlimb twitches was 0.72 mA (0.4–1.0 mA). Lowest mean (range) current to elicit tail twitches was 0.58 mA (0.4–1.0 mA). Tail twitches were reliably lost at mean current of 0.37 mA (0.2–0.8). Epidural anesthesia was considered to be successful in 19/20 dogs. In only 9/20 dogs, needle placement would have been correct based on using ‘classic’ indicators alone (‘pop’ as enter epidural space, loss of resistance to injection). The results of this study suggest that nerve stimulation may be useful in confirming correct epidural needle placement prior to drug administration.