No abstract

Accurate pain assessment methods are necessary to ensure animal welfare and reliable data collection in animal research.The Rat Grimace Scale (RGS), a facial expression pain scale, allows effective identification of pain. However, the potentialconfounds of this method remain mostly unexplored. General anesthesia, which is used in many laboratory procedures,suppresses thermoregulation and results in hypothermia. We investigated the effects of isoflurane-induced hypothermia onRGS scores. Twenty (10 male and 10 female) Sprague–Dawley rats each received 30 min of anesthesia, followed by 30 minof observation after the return of sternal recumbency. Rats were randomized to receive warming with an electric heatingpad or no warming during both periods. Unwarmed rats became hypothermic within 15 min after isoflurane exposure beganand returned to normothermia within 15 min after returning to sternal recumbency. Warmed rats did not deviate from thenormothermic range. The RGS scores of unwarmed rats were significantly higher than baseline levels for 3 h after anesthesiaand were higher than those of warmed rats at 5 and 180 min after anesthesia. Hypothermia resulted in a larger proportion ofrats crossing a predetermined analgesic intervention threshold. Our findings show that hypothermia induced by isofluraneanesthesia presents a confound to accurate RGS scoring. These results emphasize the importance of maintaining normothermiato avoid inflated pain scores and to obtain accurate pain assessment.

Spermatogonial stem cells (SSC) are the foundation of spermatogenesis. Undifferentiated spermatogonia, containing SSC, represent only 2 to 5% of cells recovered from immature mammalian testis. Cryopreservation in liquid nitrogen allows for long-term storage of cells. Preservation of germ cells can serve as a means of genetic preservation from immature males when sperm storage is not an option. Studies have investigated the effects of cryopreservation on the spermatogenic potential of SSC and the efficiency of various cryopreservation protocols. Preliminary observations indicated that germ cells may survive cryopreservation better than testicular somatic cells, resulting in a post-thaw cell population enriched in germ cells. However, this has not been critically evaluated. The objective of this study was to test the hypothesis that germ cells are less susceptible to cryo-damage than testicular somatic cells. Cells were harvested from the testes of 1-wk-old piglets by 2-step enzymatic digestion. The initial cell suspension was subjected to differential adhesion to enrich the cell population for germ cells. Cells were plated in DMEM + 5% fetal bovine serum and incubated at 37°C in 5% CO2 in air. After 18 h, cells in suspension and cells slightly attached were recovered by trypsinization (1 : 10 trypsin-ethylenediaminetetraacetic acid) for 30 s and replated. This was repeated 24 and 36 h after initial plating. The enriched population was placed into cryovials at a concentration of 30 × 106 cells in freezing media (70% DMEM + 20% fetal bovine serum + 10% dimethyl sulfoxide), kept for 24 h at –80°C in a cryogenic freezing container and transferred to liquid nitrogen for 1 week. Aliquots of cells before freezing and after thawing at 37°C followed by incubation at 37°C in 5% CO2 in air for 1 h were analyzed for viability by propidium iodide (PI) exclusion and immunofluorescence for the germ cell marker VASA to identify viable germ cells (VASA+/PI–), nonviable germ cells (VASA+/PI+), viable somatic cells (VASA–/PI–), and nonviable somatic cells (VASA–/PI+). The percentage of viable germ cells after freezing and thawing was compared to the percentage of viable somatic cells by ANOVA. After enrichment by differential plating, the cell population had 95.6 ± 0.9% viability and contained 27.1 ± 7.4% germ cells (n = 3 replicates). After cryopreservation, the overall cell viability was 77.5 ± 1.6%, and 25.8 ± 8.0% were germ cells. The overall viability after cryopreservation could potentially have benefited from the 1-h incubation prior to analysis. The viability of the germ cell population after freezing and thawing was higher (92.1 ± 3.1%) than somatic cell viability (72.3 ± 1.7%; P < 0.01). These results indicate that porcine germ cells survive cryopreservation better than do testicular somatic cells. Therefore, cryostorage of germ cells can be an efficient means for preservation of male genetic material. Supported by NIH ORIP/DCM RR17359.