OBJECTIVE To develop a high-resolution melting (HRM) assay to detect the g.66493737C>T polymorphism in the myostatin gene (MSTN) and determine the frequency of 3 previously defined g.66493737 genotypes (T/T, T/C, and C/C) in warmblood horses.
SAMPLES Blood samples from 23 horses.
PROCEDURES From each blood sample, DNA was extracted and analyzed by standard PCR methods and an HRM assay to determine the MSTN genotype. Three protocols (standard protocol, protocol in which a high-salt solution was added to the reaction mixture before the first melting cycle, and protocol in which an unlabeled probe was added to the reaction mixture before analysis) for the HRM assay were designed and compared. Genotype results determined by the HRM protocol that generated the most consistent melting curves were compared with those determined by sequencing.
RESULTS The HRM protocol in which an unlabeled probe was added to the reaction mixture generated the most consistent melting curves. The genotypes of the g.66493737C>T polymorphism were determined for 22 horses (16 by HRM analysis and 20 by sequencing); 14, 7, and 1 had the T/T, T/C, and C/C genotypes, respectively. The genotype determined by HRM analysis agreed with that determined by sequencing for 14 of 16 horses. The frequency of alleles T and C was 79.5% and 20.5%, respectively.
CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that HRM analysis may be a faster and more economical alternative than PCR methods for genotyping. Genotyping results might be useful as predictors of athletic performance for horses.
To compare stress markers, gastrointestinal motility, and behavioral indicators of pain between guinea pigs undergoing pneumoperitoneum with carbon dioxide (CO2) and control guinea pigs.
Fourteen 4- to 5-month-old intact female Hartley guinea pigs.
Guinea pigs were randomized to receive insufflation or serve as controls (anesthesia and abdominal catheter placement without insufflation), with 7 animals/group. Insufflated animals underwent 6 mm Hg of CO2 pneumoperitoneum for 30 minutes. Afterward, results for vital signs, blood glucose, fecal cortisol, appetite, fecal output, and behaviors (via video recording) were compared between the 2 groups.
There was no difference between groups and over time for body temperature, heart rate, fecal output in grams, pellets consumed, blood glucose, and fecal cortisol. Guinea pigs that underwent insufflation had significantly more fecal pellets at 36 hours after the procedure. Several behaviors were expressed similarly between groups and over time, such as body turns, incomplete movement, rearing, lying down, drinking, and hiding. Coprophagy occurred less often in the insufflated versus noninsufflated group at 12 h postprocedure but was similar between groups at other time points. At 60 hours after the procedure, insufflated animals spent less time squinting compared to noninsufflated animals. Other behaviors were differentially expressed over time but not between treatments.
Overall, there were no major differences in appetite, stress markers, and behaviors between insufflated and control guinea pigs. CO2 insufflation did not appear to cause undue pain or stress in guinea pigs and may be a reasonable technique to use during laparoscopy.