Proteomics approach reveals urinary markers for early pregnancy diagnosis in buffaloes.

This study aimed to compare urine proteomics from non- and pregnant buffaloes in order to identify potential biomarkers of early pregnancy. Forty-four females underwent hormonal ovulation synchronization and were randomly divided into two experimental groups: inseminated (n = 30) and non-inseminated (n = 14). The pregnant females were further divided into two groups: pregnant at Day 12 (P12; n = 8) and at Day 18 (P18; n = 8) post-ovulation. The non-pregnant group was also subdivided into two groups: non-pregnant at Day 12 (NP12; n = 7) and at Day 18 (NP18; n = 7). Urine was collected from all females on Days 12 or 18. The samples were processed for proteomics. A total of 798 proteins were reported in the urine considering all groups. The differential proteins play essential roles during pregnancy, acting in cellular transport and metabolism, endometrial remodeling, embryonic protection, and degradation of defective proteins. We suggest that some proteins from our study can be considered biomarkers for early pregnancy diagnosis, since they were increased in pregnant buffaloes. SIGNIFICANCE: Macromolecules have been studied for early pregnancy diagnosis, aiming to increase reproductive efficiency in cattle and buffaloes. Direct methods such as rectal palpation and ultrasonography have been considered late. Thus, this study aimed to compare urine proteomics from non- and pregnant buffaloes to identify potential biomarkers of early pregnancy. The differential proteins found in our study play essential roles during pregnancy, acting in cellular transport and metabolism, endometrial remodeling, embryonic protection, and degradation of defective proteins. We suggest that these proteins can be considered possible biomarkers for early pregnancy diagnosis since they were increased in the pregnant buffaloes.

First successful frozen semen of the maned wolf (Chrysocyon brachyurus).

This study aimed to describe successful cryopreservation of sperm from maned wolves (Chrysocyon brachyurus). Three ejaculates from 2 maned wolves were collected by digital manipulation of the penis and evaluated subjectively, centrifuged and frozen in BotuCrio® (Botupharma, Botucatu, Brazil) or Tris-yolk egg extender. Spermatozoa were thawed at 37ºC/30s or 70ºC/4s and evaluated for kinetics, morphology, plasma and acrosome membrane integrity, mitochondrial potential, hydrogen peroxide, superoxide anion and lipid peroxidation. From 5 thawed samples, two had sperm total motility >55% (56.0% and 64.0%) and progressive motility ~35% (35% and 40%), both frozen with Tris-yolk egg. Plasma and acrosome membrane integrity decreased and percentage of sperm defects increased post-thawing. We concluded that is possible to freeze spermatozoa from maned wolves using semen collection and processing methods applied for domestic dogs.

Proteomics Approach of Rapamycin Anti-Tumoral Effect on Primary and Metastatic Canine Mammary Tumor Cells In Vitro.

Rapamycin is an antifungal drug with antitumor activity and acts inhibiting the mTOR complex. Due to drug antitumor potential, the aim of this study was to evaluate its effect on a preclinical model of primary mammary gland tumors and their metastases from female dogs. Four cell lines from our cell bank, two from primary canine mammary tumors (UNESP-CM1, UNESP-CM60) and two metastases (UNESP-MM1, and UNESP-MM4) were cultured in vitro and investigated for rapamycin IC50. Then, cell lines were treated with rapamycin IC50 dose and mRNA and protein were extracted in treated and non-treated cells to perform AKT, mTOR, PTEN and 4EBP1 gene expression and global proteomics by mass spectrometry. MTT assay demonstrated rapamycin IC50 dose for all different tumor cells between 2 and 10 µM. RT-qPCR from cultured cells, control versus treated group and primary tumor cells versus metastatic tumor cells, did not shown statistical differences. In proteomics were found 273 proteins in all groups, and after data normalization 49 and 92 proteins were used for statistical analysis for comparisons between control versus rapamycin treatment groups, and metastasis versus primary tumor versus metastasis rapamycin versus primary tumor rapamycin, respectively. Considering the two statistical analysis, four proteins, phosphoglycerate mutase, malate dehydrogenase, l-lactate dehydrogenase and nucleolin were found in decreased abundance in the rapamycin group and they are related with cellular metabolic processes and enhanced tumor malignant behavior. Two proteins, dihydrolipoamide dehydrogenase and superoxide dismutase, also related with metabolic processes, were found in higher abundance in rapamycin group and are associated with apoptosis. The results suggested that rapamycin was able to inhibit cell growth of mammary gland tumor and metastatic tumors cells in vitro, however, concentrations needed to reach the IC50 were higher when compared to other studies.