Simultaneous steroids measurement in dogs with hyperadrenocorticism using a column-switching liquid chromatography-tandem mass spectrometry method

We developed an analytical method using an on-line column-switching liquid chromatography with triple quadrupole mass spectrometry (LC/MS/MS) for quantifying multiple steroids in serum. Using the developed method, we evaluated the serum concentration of nine steroids (cortisol, corticosterone, cortisone, 11-deoxycortisol, 21-deoxycortisol, deoxycorticosterone, progesterone, 17α-OH-progesterone and aldosterone) in dogs with hyperadrenocorticism (HAC). Serum was mixed with stable isotope internal standards and thereafter purified by the automated column-switching system. The limit of detection ranged 2–16 pg/ml for nine steroids. In the baseline samples, five steroids (cortisol, corticosterone, cortisone, 11-deoxycortisol, and 17α-OH-progesterone) were detected in all dogs. The concentrations of cortisone, 11-deoxycortisol, and 17α-OH-progesterone in dogs with HAC (n=19) were significantly higher those in dogs without HAC (n=15, P<0.02). After the adrenocorticotropic hormone stimulation test, six steroids (cortisol, corticosterone, cortisone, 11-deoxycortisol, 17α-OH-progesterone, and deoxycorticosterone) were above the limit of quantification in all dogs. Cortisol, corticosterone, cortisone, and deoxycorticosterone concentrations of dogs with HAC were significantly higher than those of dogs without HAC (P<0.02). In addition, 11-deoxycortisol and 17α-OH-progesterone concentration was higher in dogs with HAC than in dogs without HAC (P=0.044 and P=0.048, respectively). The on-line column-switching LC/MS/MS would be feasible for measuring multiple steroids in dog serum. The results suggest that cortisone, 11-deoxycortisol, and 17α-OH-progesterone would be related to HAC. Further studies are warranted to assess the clinical feasibility of steroid profile in dogs with HAC.     

Participation of phosphofructokinase,malate dehydrogenase and isocitrate dehydrogenase in capacitation and acrosome reaction of boar spermatozoa

The aim of this work was to determine the enzymatic activity of phosphofructokinase (PFK), malate dehydrogenase (MDH) and isocitrate dehydrogenase (IDH) in boar spermatozoa and study their participation in bicarbonate‐induced capacitation and follicular fluid‐induced acrosome reaction. Enzymatic activity of these enzymes was determined spectrophotometrically in extracts of boar spermatozoa. Sperm suspensions were incubated in the presence of bicarbonate (40 mM), a well‐known capacitation inducer, or follicular fluid (30%), as an acrosome reaction inducer, and different concentrations of oxoglutarate, oxalomalate and hydroxymalonate, inhibitors of PFK, IDH and MDH, respectively. Capacitation percentages were determined by the fluorescence technique of chlortetracycline (CTC), and true acrosome reaction was determined by trypan blue and differential–interferential contrast, optical microscopy. The activity of PFK in boar spermatozoa enzymatic extracts was 1.70 ± 0.19 U/10¹⁰ spermatozoa, the activity of NAD‐ and NADP‐dependent IDH was 0.111 ± 0.005 U/10¹⁰ and 2.22 ± 0.14 U/10¹⁰ spermatozoa, respectively, and the activity of MDH was 4.24 ± 0.38 U/10¹⁰ spermatozoa. The addition of the specific inhibitors of these enzymes prevented sperm capacitation and decreased sperm motility during capacitation and inhibited the acrosome reaction (AR), without affecting the sperm motility during this process. Our results demonstrate the participation of PFK, IDH and MDH in bicarbonate‐induced capacitation and follicular fluid‐induced acrosome reaction in boar spermatozoa, contributing to elucidate the mechanisms that produce energy necessary for these processes in porcine spermatozoa.     

Comparison of Antral and Preantral Ovarian Follicle Populations Between Bos indicus and Bos indicus‐taurus Cows with High or Low Antral Follicles Counts

The objective was to compare populations of antral and pre‐antral ovarian follicles in Bos indicus and Bos indicus‐taurus cows with high and low antral follicle counts. Nelore (Bos indicus, n = 20) and Nelore X Angus (1/2 Bos indicus‐taurus, n = 20) cows were subjected to follicular aspiration without regard to the stage of their oestrous cycle (day of aspiration = D0) to remove all follicles ≥3 mm and induce growth of a new follicular wave. Ovaries were examined by ultrasonography on D4, D19, D34, D49 and D64, and antral follicles ≥3 mm were counted. Thereafter, cows were assigned to one of two groups: high or low antral follicular count (AFC, ≥30 and ≤15 antral follicles, respectively). After D64, ovaries were collected after slaughter and processed for histological evaluation. There was high repeatability in the numbers of antral follicles for all groups (range 0.77–0.96). The mean (±SD) numbers of antral follicles were 35 ± 9 (Bos indicus) and 38 ± 6 (Bos indicus‐taurus) for the high AFC group and 10 ± 3 (Bos indicus) and 12 ± 2 (Bos indicus‐taurus) follicles for the low AFC. The mean number of preantral follicles in the ovaries of Bos indicus‐taurus cows with high AFC (116 226 ± 83 156 follicles) was greater (p < 0.05) than that of Bos indicus cows (63 032 ± 58 705 follicles) with high AFC. However, there was no significant correlation between numbers of antral and preantral follicles.     

Fusion of Boar Sperm with Nanoliposomes Prepared from Synthetic Phospholipids

Liposomes are artificial membrane vesicles that can be used to test and model the functions and interactions of various biological membranes, or as a carrier system to deliver biologically active substances into the cells, or to incorporate lipids into the plasma membrane of target cells to modify membrane structure–function relationships. Sperm plasma membrane undergoes lipid modification during maturation in epididymis and during capacitation in the female reproductive tract to facilitate fertilization. Natural variation in the amounts and composition of lipids in the sperm plasma membrane may also contribute to the species‐specific sperm sensitivities to handling and storage conditions. Boar sperm are notoriously susceptible to membrane damage and are resistant to compositional alteration by artificial liposomes. This study used flow cytometry to demonstrate stable incorporation of nanoliposomes prepared from a complex mixture of various phospholipids (phosphatidylcholine : phosphatidylethanolamine : sphingomyelin : phosphatidylserine : phosphatidylinositol) with high fusion efficiency. Over 90% of sperm rapidly took up fluorescently labelled liposomes and retained the lipids for at least 60 min, in a significant time‐ and concentration‐dependent manner. This unique fusion efficacy could be used to alter sperm plasma membrane composition and hence membrane‐based functional responses.     

Effect of mitochondrial uncoupling and glycolysis inhibition on ram sperm functionality

Studies have demonstrated the importance of mitochondria to sperm functionality, as the main source of ATP for cellular homoeostasis and motility. However, the role of mitochondria on sperm metabolism is still controversial. Studies indicate that, for some species, glycolysis may be the main mechanism for sperm energy production. For ram sperm, such pathway is not clear. Thus, we evaluated ram sperm in response to mitochondrial uncoupling and glycolysis inhibition aiming to assess the importance of each pathway for sperm functionality. Statistical analysis was performed by the SAS System for Windows, using the General Linear Model Procedure. Data were tested for residue normality and variance homogeneity. A p < .05 was considered significant. Groups treated with the mitochondrial uncoupler Carbonyl cyanide 3 chlorophenylhydrazone (CCCP) showed a decrease in the percentage of cells with low mitochondrial activity and high mitochondrial membrane potential. We also observed that the highest CCCP concentration promotes a decrease in sperm susceptibility to lipid peroxidation. Regardless the lack of effect of CCCP on total motility, this substance induced significant alterations on sperm kinetics. Besides the interference of CCCP on spermatic movement patterns, it was also possible to observe such an effect in samples treated with the inhibitor of glycolysis (2‐deoxy‐d ‐glucose, DOG). Furthermore, treatment with DOG also led to a dose‐dependent increase in sperm susceptibility to lipid peroxidation. Based on our results, we suggest that the glycolysis appears to be as important as oxidative phosphorylation for ovine sperm kinetics as this mechanism is capable of maintaining full motility when most of the cells have a low mitochondrial membrane potential. Furthermore, we found that changes in the glycolytic pathway trough glycolysis inhibition are likely involved in mitochondrial dysfunction and sperm oxidative unbalance.     

Relation between respiratory activity and sperm parameters in boar spermatozoa cryopreserved with alpha‐tocopherol and selected by Sephadex

Our aim was to evaluate the effect of Sephadex filtration on respiratory activity of porcine spermatozoa and its relation with quality and functional sperm parameters. Samples were evaluated regarding oxygen uptake and sperm parameters: motility, plasma and acrosome membrane integrity, capacitation and acrosome reaction induction in vitro, plasma membrane functionality, determined by the hypo‐osmotic swelling test (HOST), and lipid peroxidation assessed by thiobarbituric acid assay. Sephadex filtration improved all routine quality parameters (motility, plasma and acrosome membrane integrity) and functional parameters (HOST, in vitro capacitation and true acrosome reaction levels) and produced a significant decrease in cryocapacitation and lipid peroxidation. Oxygen uptake increased in Sephadex samples (41 ± 7%) respect to single washing. Oxygen addition of carbonyl‐cyanide‐m‐chlorophenylhydrazone (CCCP) confirmed mitochondrial coupling in washed and Sephadex samples; showing an increase of 2.6 and 4.2 times for oxygen consumption in single washing and Sephadex ones, respectively. The increase in oxygen uptake with succinate addition with respect to basal oxygen uptake was significantly lower in Sephadex samples (63 ± 25%) than in the washed ones (183 ± 35%). Sephadex samples showed higher mitochondrial activity measured by oxygen consumption and improved quality and functional parameters. Our study recommends this protocol due to the fact that this filtration method removes dead or damaged spermatozoa allowing to obtain cryopreserved boar spermatozoa with optimized fertilizing capacity.