Assessment of Vcheck® analyzer for rapid progesterone concentration measurement including recommendations for achieving the optimal breeding time in bitches.

Background and Aim: Serum progesterone concentration plays critical role in determining the optimal breeding time in bitches and diagnosing reproductive-related issues. This study aimed to conduct a comparative analysis of serum progesterone results obtained from commercial point-of-care immunological analyzers, namely, Vcheck®, with those obtained using chemiluminescent microparticle immunoassay (CMIA). Our overarching goal was to evaluate these analyzers' accuracy and establish standardized guidelines for optimal breeding timing. Materials and Methods: Ninety-four serum samples from bitches were analyzed using the Vcheck® analyzer and compared with CMIA. Thorough documentation included the mean, standard deviation, 95% confidence interval (CI), and minimum and maximum values of serum progesterone concentrations. Furthermore, Pearson's correlation coefficient, Lin's concordance correlation coefficient, and the bias correction factor were meticulously recorded. Results: The mean progesterone concentration measured using the Vcheck® analyzer was significantly lower than that measured using CMIA, with a mean difference of 1.26 ng/mL of serum. The Bias correction factor was 0.935, which was nearly 1.00, indicating that the line of best-fit was on the perfect line of agreement, providing insight into the measurement accuracy. Pearson's correlation coefficient, a measure of precision, was also close to 1 (0.939), confirming the reliability of the data. Furthermore, Lin's concordance correlation coefficient was 0.877, indicating a fair overall agreement between the Vcheck® and CMIA methods. These results support the validity of the Vcheck® analyzer's results. The present study was developed by aligning with established CMIA guidelines and adapting them using the range and 95% CI derived from each set of results, ensuring a standardized and rigorous approach. Conclusion: The Vcheck® analyzer provides a rapid assessment of serum progesterone concentration in bitches, with results comparable to those measured using the CMIA technique. However, when considering the use of the Vcheck® analyzer, it is recommended that the results should be interpreted carefully and the interpretation guidelines should be followed. In conclusion, Vcheck® provides a reliable and convenient method for veterinarian practitioners to measure canine progesterone levels in a clinical/hospital setting.

A Precision Assessment of a Point-of-Care Immunological Analyzer for Swift Progesterone Measurement and Guidance for Determining the Optimal Breeding Time in Bitches.

The measurement of serum progesterone often varies due to different laboratory methodologies and individual canine characteristics. In this investigation, serum progesterone outcomes obtained from a commercial point-of-care immunological analyzer, designed for efficient serum progesterone assessment in bitches, were compared with results derived from chemiluminescent microparticle immunoassay from reference laboratories in Thailand. Our thorough documentation encompassed various parameters: mean, standard deviation, 95% confidence interval, and minimum and maximum serum progesterone concentration values. Additionally, we meticulously recorded the Pearson's correlation coefficient, Lin's concordance correlation coefficient, and the bias correction factor. Interestingly, there was no significant difference (p > 0.05) in the means obtained by the point-of-care immunological analyzer and chemiluminescent microparticle immunoassay. The Pearson's correlation coefficient between the point-of-care immunological analyzer and chemiluminescent microparticle immunoassay stood at 0.957, with Lin's concordance correlation coefficient for point-of-care immunological analyzer recorded as 0.949. Furthermore, the bias correction factor was established at 0.991. This investigation followed established chemiluminescent microparticle immunoassay guidelines, modified to incorporate the mean and 95% confidence interval as criteria for optimal breeding time using the point-of-care immunological analyzer. In conclusion, the commercial point-of-care immunological analyzer emerges as a valuable tool, aiding in precisely determining the optimal timing for natural mating or artificial insemination in bitches.

Membrane-permeable trehalose improves the freezing ability and developmental competence of in-vitro matured feline oocytes.

Oocytes are highly sensitive to cryopreservation, which frequently results in an irreversible loss of developmental competence. We examined the effect of membrane-permeable trehalose on the freezing ability of feline oocytes matured in vitro. In Experiment 1, intracellular trehalose (trehalose hexaacetate; Tre-(OAc)6) was synthesized from trehalose precursor and subjected to spectroscopic characterization. The membrane permeability of the Tre-(OAc)6 was investigated by incubating oocytes with different concentrations of Tre-(OAc)6 (3, 15, and 30 mM). Optimum concentration and the toxicity of Tre-(OAc)6 were assessed in Experiment 2. The effects of Tre-(OAc)6 on freezing ability in terms of apoptotic gene expression and developmental competence of in-vitro matured oocytes were examined in Experiments 3 and 4, respectively. The Tre-(OAc)6 permeated into the ooplasm of cat oocytes in a dose- and time-dependent manner. The highest concentration of intracellular trehalose was detected when the oocytes were incubated for 24 h with 30 mM Tre-(OAc)6. For the toxicity test, incubation of oocytes with 3 mM Tre-(OAc)6 for 24 h did not affect maturation rate and embryo development. However, high doses of Tre-(OAc)6 (15 and 30 mM) significantly reduced maturation and fertilization rates (p < 0.05). In addition, frozen-thawed oocytes treated with 3 mM Tre-(OAc)6 significantly upregulated anti-apoptotic (BCL-2) gene expression compared with the control (0 mM) and other Tre-(OAc)6 concentrations (15 and 30 mM). Oocyte maturation in the presence of 3 mM Tre-(OAc)6 prior to cryopreservation significantly improved oocyte developmental competence in terms of cleavage and blastocyst rates when compared with the control group (p < 0.05). Our results lead us to infer that increasing the levels of intracellular trehalose by Tre-(OAc)6 during oocyte maturation improves the freezing ability of feline oocytes, albeit at specific concentrations.

Effects of vitrification and a Rho-associated coiled-coil containing protein kinase 1 inhibitor on the meiotic and developmental competence of feline oocytes.

Oocyte cryopreservation is the technique of choice for the long-term storage of female gametes. However, it induces an irreversible loss of oocyte viability and function. We examined the effects of vitrification and a Rho-associated coiled-coil containing protein kinase 1 (ROCK1) inhibitor (ROCKi) on the meiotic and developmental competence of feline oocytes. We examined the expression of LIM kinase (LIMK) 1 and 2, with and without ROCKi treatment. Cumulus oocyte complexes (COCs) were matured in vitro with 0, 10, 20, and 40 µM ROCKi. The oocytes were subsequently assessed for maturation rate and embryo development following in vitro fertilization. We repeated the COC experiment, but vitrified and warmed the COCs prior to culture. We detected LIMK1 and LIMK2 expression in feline oocytes, which could be downregulated by ROCKi treatment. The ROCKi at 10 µM affected neither meiotic nor developmental competence (P > 0.05, versus control). However, high concentrations of ROCKi during maturation induced meiotic arrest at metaphase I. Appropriate concentrations of ROCKi significantly improved the normal fertilization rate of vitrified warmed oocytes (49.4 ± 3.4%) compared with that of the control (42.8 ± 8.6%, P < 0.05). The ROCKi also significantly improved the embryo cleavage rate (36.1 ± 3.8%) as compared with the non-treated control (27.4 ± 2.5%, P < 0.05). Thus, this study revealed that the main mediators of the ROCK cascade (LIM kinases) are expressed in feline oocytes. The ROCKi (10 µM) did not compromise the meiotic or developmental competence of feline oocytes. In addition, 10 µM ROCKi improved the cytoplasmic maturation of vitrified-warmed oocytes as indicated by their fertilization competence.