Evaluation of DNA integrity and chromatin condensation in cat sperm collected by urethral catheterization and epididymis slicing.

The aim of this study was to evaluate the effectiveness of the original sperm chromatin dispersion (SCD) assay and the toluidine blue (TB) stain to assess DNA fragmentation and chromatin condensation, respectively, in cat sperm obtained by urethral catheterization (CT) and epididymis slicing (EP). CT and EP samples were collected from the same cat, and sperm motility, concentration, morphology, DNA integrity and chromatin condensation were evaluated. As controls, aliquots of the samples were incubated with 0.3 M NaOH and with 1% of dithiothreitol (DTT) to promote DNA fragmentation and chromatin decondensation, respectively. With SCD, four DNA dispersion halo patterns were observed: large, medium, small and no halo. TB staining patterns were as follows: light blue (condensed chromatin), light violet (moderate chromatin decondensation) and dark blue-violet (high chromatin decondensation). Sperm incubations with NaOH and with DTT were effective in inducing DNA fragmentation and chromatin decondensation, respectively. No significant differences were observed in the percentages of the SCD and TB patterns between samples (CT and EP) and no correlation was observed between sperm head abnormalities and the different SCD and TB patterns. The original SCD technique and the TB stain were adapted to evaluate DNA integrity and chromatin condensation in cat sperm obtained by CT and EP.

Effect of early administration of progesterone on the function of the corpus luteum of llamas.

The aim of the present study was to evaluate if early administration of progesterone immediately after ovulation affects corpus luteum lifespan in llamas. Female llamas (n = 16) were induced to ovulate by Buserelin injection in the presence of an ovulatory follicle (Day 0). On Day 2, ovulation was confirmed and animals were randomly divided into two groups: treated animals (n = 8) received an intravaginal device containing 0.3 g of progesterone from Day 2 to Day 6 post-induction of ovulation and control group (n = 8) received a device with 0 g of progesterone. Blood samples were collected daily to determine plasma progesterone concentration and transrectal ultrasonographies were performed from Day 7 to Day 12 post-induction of ovulation. Mean maximum diameter of the corpus luteum was significantly lower and was reached before in the treated group than in the control group. The mean highest plasma progesterone concentration and the day that concentration was achieved were similar between groups. However, mean plasma progesterone concentration was significantly higher in the treated group than in the control group on Days 3 and 4 and lower on Days 8 and 9 post-induction of ovulation. The day that plasma progesterone concentration returns to 1 ng/ml differed between groups, occurring earlier in the treated group. In conclusion, the early increase of plasma progesterone concentration during the luteal phase, promoted the premature activation of the luteolytic process affecting corpus luteum function in llamas as it was previously reported in other species.

A feasible method for llama embryo paraffin embedding.

Maternal recognition of pregnancy in llamas would be related to a still-unknown signal secreted by the embryo to extend the corpus luteum lifespan. In order to study llamas reproductive physiology and early pregnancy aspects, research on embryos between 8 and 12 days post-mating is essential. However, there is a lack of information on llama embryo paraffin embedding, a procedure that exhibits difficulties, mostly for embryo manipulation. This work presents an accessible, easy and rapid protocol for embedding llama blastocysts. Uterine flushings from four superovulated llamas were performed to recover embryos 8 days post-mating. Each embryo was fixed in 1 ml of 10 % neutral buffered formalin for 24 h, at 4 °C, and transferred to 70 % ethanol. Later, they were submitted to a series of baths: 500 µl PBS with 5 % bovine foetal serum solution (PBS+BFS) for 1 min; zona pellucida permeabilization with 500 µl PBS with 0.2 % Triton X-100 for 20 min; 500 µl PBS+BFS for 1 min; dehydration on baths with ascendent grades of ethanol (70 %, 96 %, and 100 % twice) for 10 min each; and bath of butanol for 5 min. Finally, embryos were embedded in a pellet of paraffin and afterwards in a paraffin block. Complete sections from 8 of 13 grade I and II embryos were obtained. In conclusion, by applying this method, good quality sections with preserved embryo morphology suitable for histological, histochemical or immunohistochemical approaches would be possible to acquire.

Evaluation of DNA fragmentation in dog sperm using the sperm chromatin dispersion test.

The study's objective was to adapt the Sperm Chromatin Dispersion (SCD) protocol to evaluate sperm DNA fragmentation and implement a fragmentation control in dogs. Correlation between DNA status and routine sperm parameters was also analysed. To adapt the SCD, two different mercaptoethanol (ME) concentrations were assayed (2.5% and 5%) in fourteen ejaculates from seven dogs and semen incubation with 0.3 M NaOH for 15 min at room temperature was assayed as a control for sperm DNA fragmentation. Data were analysed using a Mann-Whitney test and either Pearson's or Spearman's correlation. The selected ME concentration to use in the SCD test was 5%, as it produced the largest DNA dispersion halo while preserving the core nucleus structure. Four DNA halo patterns were identified as follows: large dispersion halos, medium halos, small halos and nuclei without halos. Semen incubated with NaOH showed 100% sperm without halos (damaged DNA). A significant positive correlation was observed between sperm with fragmented DNA and sperm with coiled tails. Thus, it was possible to adapt the SCD protocol to evaluate dog sperm DNA fragmentation in raw semen without using a commercial kit and establish incubation with NaOH as a DNA fragmentation control. Only coiled tails showed correlation with DNA fragmentation.

Corpus luteum vascularization during the maternal recognition of pregnancy in llamas (Lama glama).

The aim of this study was to characterize corpus luteum vascularization and its association with plasma progesterone concentration in early stages of pregnancy, when maternal recognition of pregnancy is expected to occur. In all animals, both plasma progesterone concentration and corpus luteum vascularization increased from Day 6 to Day 8 post-mating and afterwards in non-pregnant llamas they started to decrease to reach basal levels around Days 12 to 14 post-mating, while in pregnant animals, both variables remained elevated until the end of the study. A lineal positive relationship between corpus luteum vascularization and plasma progesterone concentration was observed in pregnant (r2  = .46, p < .0001) and non-pregnant llamas (r2  = .66, p < .0001). Pregnant animals showed higher plasma progesterone concentration and corpus luteum vascularization than the non-pregnant ones from Day 12 post-mating until the end of the study (p Ë‚ .05 and p Ë‚ .01, respectively). These results suggest that maternal recognition of pregnancy should occur before Day 12 post-mating in order to expand luteal lifespan, maintaining corpus luteum vascularization and progesterone production. Also, the assessment of CL vascularization area could be a useful and non-invasive method for early pregnancy diagnosis due to its association with plasma progesterone concentration.

Overexpression of 11ß-hydroxysteroid dehydrogenase 1 in visceral adipose tissue and underexpression of endothelial nitric oxide synthase in the adrenal cortex of dogs with hyperadrenocorticism.

11ß-Hydroxysteroid dehydrogenase 1 (11ß-HSD1) is an enzyme that activates cortisone into cortisol in tissues. Alterations in this enzyme are related to the development of metabolic syndrome, obesity and hyperadrenocorticism (HAC). Endothelial nitric oxide synthase (eNOS) produces nitric oxide and is related to the regulation of adrenal steroidogenesis. The aim of the study was to evaluate 11ß-HSD1 and eNOS expression in dogs with HAC. Visceral adipose tissue samples were taken to evaluate 11ß-HSD1 expression by immunohistochemistry and western blotting. In parallel, adrenal gland samples were collected to evaluate eNOS expression by immunohistochemistry. 11ß-HSD1 expression was significantly higher in the adipocytes of dogs with HAC than in those of the control dogs. eNOS expression in the adrenal cortex (zona fasciculata) was significantly lower in the dogs with HAC than in the control dogs. 11ß-HSD1 overexpression and eNOS underexpression could play a role in the maintenance of hypercortisolism in dogs with HAC.

Effect of SOM230 (pasireotide) on corticotropic cells: action in dogs with Cushing's disease.

SOM230 (pasireotide) is a multiligand somatostatin (SRIF) analog able to bind to somatostatin receptor (SSTR) subtypes 1, 2, 3 and 5, and trigger antisecretory and antiproliferative signaling cascades. Canines have become in vivo models to test the pharmacological treatment of corticotropinomas because they frequently develop Cushing's disease in a spontaneous manner, due to adrenocorticotropic hormone (ACTH)-producing pituitary adenomas. Different levels of expression of SSTR2 and SSTR5 have been shown in both mouse AtT20 cells and canine tumoral corticotropinoma cells. The objective of this study was to evaluate whether SOM230 controls both tumor cell growth and hormone synthesis, therefore controlling the disease. SOM230 was tested in dogs suffering from Cushing's disease (10 animals were treated continuously during 6 months, and another 10 were treated with 3 cycles consisting of 2 months of treatment followed by a 2-month rest period). A significant decrease in ACTH, urinary cortisol creatinine ratio, adenoma size (magnetic nuclear resonance) and improvement of clinical signs were obtained, without side effects. AtT20 cells treated with SOM230 suppressed pro-opiomelanocortin (POMC) promoter activity through SSTR2, via the G(i) α-subunit, and reduced Nur77/Nurr1 transcriptional activity. We conclude that SOM230, in addition to its well-described antisecretory effects, inhibits, as shown in AtT20 cells, ACTH synthesis at the POMC transcriptional level, an effect mediated mainly through SSTR2, and limits tumor growth. The controlled Cushing's disease in the dogs that received the treatment indicates that SOM230 has a potential therapeutic use in humans suffering from Cushing's disease.