Haralabos N Ververidis

Effect of antioxidant supplementation in semen extenders on semen quality and reactive oxygen species of chilled canine spermatozoa.

The objective of this study was to evaluate quality of chilled dog semen processed with extenders containing various antioxidants. Single ejaculates from five dogs were always pooled and evaluated for concentration, sperm motility, progressive motility (RSF-movement), viability, acrosomal integrity and by the hypo-osmotic swelling (HOS)-test. Also, superoxide (O(2)(-)) production, hydroxyl radicals (OH) and total reactive oxygen species (tROS) were determined. Pooled semen was divided in seven aliquots (for control and test conditions), which were diluted to a final concentration of 67x10(6)spermatozoa/ml with TRIS-glucose-egg yolk extender with or without the following supplements: control (without antioxidants), vitamin C (0.5mM), N-acetyl-l-cysteine (NAC; 0.5mM), taurine (0.2mM), catalase (100u/ml), vitamin E (0.1mM) and 5-(4-dimethylamino-phenyl)-2-phenyl-penta-2,4-dienoic acid (B16; 0.1mM). The semen aliquots were chilled and preserved at 4 degrees C. Portions of chilled semen were removed at 24 and 72h, and semen quality was evaluated after rewarming. At 24h the mean (+/-S.E.M.) sperm motility was higher (p<0.001) when vitamin E, taurine and B16 were added in the extender, whereas more spermatozoa with RSF-movement were observed (p<0.001) in the vitamin E, catalase, B16 and taurine groups. Sperm viability was higher (p=0.040) in B16 and vitamin E groups and the percentage of swollen spermatozoa was higher (p=0.002) only in the B16 group. Acrosomal integrity and OH were not significantly influenced by any of the antioxidants tested. Superoxide production was significantly lower when vitamin C, B16 and vitamin E were added in semen extenders compared with the control (p=0.017). All antioxidant groups, except vitamin C and NAC, contained less tROS compared to the control group, but only the B16 group value differed significantly (p=0.05). At 72h sperm motility was higher (p<0.001) when vitamin E, catalase, B16, taurine and NAC were added in the extender. More spermatozoa with RSF-movement were observed (p<0.001) in the vitamin E, catalase, B16, taurine and NAC treatment groups. Sperm viability was higher (p=0.001) when vitamin E, B16, taurine and vitamin C were added in semen extenders. HOS-test percentages were higher (p=0.016) in the B16, vitamin E, catalase and NAC groups. Acrosomal integrity was not influenced in any case. Production of O(2)(-) was significantly higher using catalase compared to all the other groups (p=0.006), while OH was not significantly influenced by any of the antioxidants tested. The addition of vitamin E, catalase and B16 in semen extenders resulted in significantly lower tROS values compared with the controls (p<0.0005). The results suggest that vitamin E and B16 had the most pronounced effect in preserving semen quality of chilled dog spermatozoa.

Effect of N‐acetyl‐L‐cysteine Supplementation in Semen Extenders on Semen Quality and Reactive Oxygen Species of Chilled Canine Spermatozoa

The objective of this study was to evaluate the quality of chilled dog semen processed with extenders containing various concentrations of N-acetyl-L-cysteine (NAC). Ejaculates from five dogs were collected, pooled and evaluated for concentration, motility, rapid steady forward movement (RSF-movement), viability, acrosomal integrity and by the hypo-osmotic swelling test (HOST). In addition, superoxide anion (O(2)(-*)) production, hydroxyl radicals (OH(*)) and total reactive oxygen species (tROS) were determined. The pool was divided into five aliquots, which were diluted to a final concentration of 66.66 x 10(6) spermatozoa/ml with Tris-glucose-egg yolk extender containing one of the following concentrations of NAC (0, 0.5, 1, 2.5 or 5 mm). The semen aliquots were chilled and preserved at 4 degrees C. Semen quality was evaluated after rewarming at 72 h. Sperm motility was significantly higher with the 0.5 mm concentration compared with the control group (p = 0.001). Rapid steady forward movement was higher with the 0.5 and 1 mm concentrations compared with the control and 5 mm group (p < 0.001). Viability and HOST percentages were not significantly altered. Compared with the control, the 5 mm concentration showed significantly reduced percentages of spermatozoa with normal acrosomes (p = 0.049). None of the ROS values at 72 h were significantly affected by the presence of NAC in semen extenders, although all NAC concentrations showed lower O(2)(-*) and OH(*) values compared with the control. Only the concentrations of 1 and 5 mm inhibited the significant increase of tROS values after 72 h, compared with the fresh semen value. In conclusion, NAC supplementation of semen extenders is beneficial to semen motility of canine spermatozoa during chilling with the 0.5 mm concentration being the most effective, although no significant ROS inhibition was observed at 72 h.

Quality and reactive oxygen species of extended canine semen after vitamin C supplementation

The objective of this study was to evaluate the quality of extended dog semen processed with diluents containing various concentrations of vitamin C. Ejaculates from five dogs were collected, pooled and evaluated for concentration, sperm motility, rapid steady forward movement (RSF-movement), viability, acrosomal integrity and by the hypo-osmotic swelling test. Also, superoxide (O(2)(-)*) production, hydroxyl radicals (OH*) and total reactive oxygen species (tROS) were determined. The pool was divided in five aliquots, which were diluted to a final concentration of 66 x 10(6) spermatozoa/ml with a Tris-glucose-egg yolk extender containing one of the following concentrations of vitamin C (0, 0.1, 0.5, 1 or 2.5 mM). The semen aliquots were chilled and preserved at 4 degrees C. Portions of chilled semen were removed at 24 and 72 h, and semen quality was evaluated after rewarming. This process was repeated 10 times in pooled semen of the same origin and data were analysed by one-way analysis of variance. At both times, none of the semen quality parameters were positively influenced (p>0.05) by vitamin C supplementation. At 24 h, none of the reactive oxygen species (O(2)(-)*, OH*, tROS) were significantly altered. At 72 h, significant reductions of O(2)(-)* production were observed by the concentrations of 0.1, 0.5 and 2.5 mM compared with the 0 mM concentration (p=0.049). Also, at 72 h, the 2.5 mM concentration showed significantly lower OH* values in comparison with the control group (p=0.048). In conclusion, addition of vitamin C to semen extenders does not benefit the quality of canine extended spermatozoa.

Use of enzyme-immunoassay for oestradiol-17β and progesterone quantification in canine serum

The objective of this investigation was to develop and evaluate competitive inhibition-enzyme-immunoassays for canine serum oestradiol-17beta (E(2)) and progesterone (P(4)) quantification. Sera from 56 healthy bitches at various stages of oestrus cycle and pregnancy were tested. For E(2) measurement, each sample (0.4 ml) was extracted with diethyl ether and after solvent evaporation the resultant hormone was reconstituted to one-fifth of the original sample volume in aqueous buffer. Each reconstitute (30 microl) was assayed for E(2) to estimate respective serum concentration. For P(4), each sample (10 microl) was directly assayed without extraction. The classic cyclic hormonal pattern during the oestrus cycle of the bitch was observed. The brief, sharp dominance of E(2) during the follicular phase was followed by the long-lasting dominance of P(4) during the luteal phase (late oestrus, dioestrus or pregnancy). During the anoestrus phase both hormones were found at basal levels, with the exception of E(2) during late anoestrus which appeared to be rising. Both assays had acceptable specificity (cross-reactions < or =10%), precision (coefficient of variation (C.V.) < 7%) and accuracy (E(2) recovery: 97%; P(4) recovery: 104.7%). The sensitivity of E(2) and P(4) assay was 4 pgml(-1) and 0.28 ngml(-1), respectively.

Effect of endogenous progesterone and oestradiol‐17β on the incidence of gastro‐oesophageal reflux and on the barrier pressure during general anaesthesia in the female dog

OBJECTIVE To investigate the potential effect of increased blood progesterone (P4) and oestradiol-17β (E2) concentrations on the barrier pressure (BrP) and the incidence of gastro-oesophageal reflux (GOR) in female dogs under general anaesthesia. STUDY DESIGN Prospective, blinded experimental trial. ANIMALS Seven female, adult, healthy dogs weighing 14-21 kg and aged 1-7 years. METHODS Each of the animals was studied under the influence of high blood E2 and basal P4 (study O), basal E2 and high P4 (study P) and basal E2 and P4 (study C) concentrations. Animals were premedicated with acepromazine and anaesthesia was induced with thiopental and maintained with halothane. Lower oesophageal pH was monitored continuously for 1 hour after induction. GOR was defined as oesophageal pH >7.5 or <4. Manometry of the posterior oesophageal sphincter (POS) was then performed using the slow pull-through technique. RESULTS Acid GOR was detected in only one animal of study O. The three studies did not differ significantly in GOR. Mean BrP was 11.2 (study O), 9.1 (study P) and 11.6 mmHg (study C). No significant differences were detected with respect to mean BrP, intra-gastric pressure and POS pressure. CONCLUSIONS It is unlikely that the increased concentrations of E2 or P4 during the normal ovarian cycle influence the functional efficiency of the POS as a major barrier to GOR in healthy, female dogs under general anaesthesia. CLINICAL RELEVANCE The fact that female dogs undergoing obstetrical surgery represented a substantial sub-group of the animals which developed postoperative benign oesophageal stricture, should probably not be attributed to the effects of increased concentrations of female sex steroid hormones.OBJECTIVE To investigate the potential effect of increased blood progesterone (P(4)) and oestradiol-17beta (E(2)) concentrations on the barrier pressure (BrP) and the incidence of gastro-oesophageal reflux (GOR) in female dogs under general anaesthesia. STUDY DESIGN Prospective, blinded experimental trial. ANIMALS Seven female, adult, healthy dogs weighing 14-21 kg and aged 1-7 years. METHODS Each of the animals was studied under the influence of high blood E(2) and basal P(4) (study O), basal E(2) and high P(4) (study P) and basal E(2) and P(4) (study C) concentrations. Animals were premedicated with acepromazine and anaesthesia was induced with thiopental and maintained with halothane. Lower oesophageal pH was monitored continuously for 1 hour after induction. GOR was defined as oesophageal pH >7.5 or <4. Manometry of the posterior oesophageal sphincter (POS) was then performed using the slow pull-through technique. RESULTS Acid GOR was detected in only one animal of study O. The three studies did not differ significantly in GOR. Mean BrP was 11.2 (study O), 9.1 (study P) and 11.6 mmHg (study C). No significant differences were detected with respect to mean BrP, intra-gastric pressure and POS pressure. CONCLUSIONS It is unlikely that the increased concentrations of E(2) or P(4) during the normal ovarian cycle influence the functional efficiency of the POS as a major barrier to GOR in healthy, female dogs under general anaesthesia. CLINICAL RELEVANCE The fact that female dogs undergoing obstetrical surgery represented a substantial sub-group of the animals which developed postoperative benign oesophageal stricture, should probably not be attributed to the effects of increased concentrations of female sex steroid hormones.

Post-partum involution of the canine uterus - gross anatomical and histological features.

We aimed to study the normal puerperium in the bitch. Ovariohysterectomy was performed in nine bitches, each at a different day after normal whelping; their genital tract was subject to gross anatomical examination, as well as to histological examination and electron microscopy scanning. Corpora albicans were evenly distributed in the left and right ovaries and placental sites were evenly distributed among left and right uterine horns. Placental sites were initially of dark green to grey colour, later becoming dark brown; their length and height progressively decreased. Height of the myometrium and diameter of the uterine glands progressively decreased. Trophoblast-like cells were consistently observed at the placental sites and on the surface of the interplacental areas, at all time points where hysterectomy had been performed. It is suggested that involution of the canine genital tract can last up to 3 months and is slow. Continuous (up to D84 post-partum) presence of prominent placental sites should be considered a normal feature of canine uterine post-partum involution.

Anesthesia and Perioperative Management of a Pneumonectomized Dog

Although left- or right-sided pneumonectomy is tolerated by normal dogs, complications impacting the respiratory, cardiovascular, and gastrointestinal systems are not uncommon. Pneumonectomy in dogs results in secondary changes in the remaining lung, which include: decreased compliance and vital capacity; and increased pulmonary vascular resistance potentially leading to right ventricular hypertrophy. Such alterations make the anesthetic management of an animal with one lung particularly challenging. This report describes a dog with a history of left pneumonectomy due to Aspergillus fumigatus pneumonia 3 yr before presentation. The dog presented with a vaginal wall prolapse, and surgical resection of the protruding vaginal wall, ovariectomy, and prophylactic gastropexy were performed. Anesthesia was induced with midazolam, fentanyl, and propofol and was maintained with isoflurane using intermittent positive pressure ventilation and a constant rate infusion of fentanyl. Epidural anesthesia was also used. Recovery and postoperative management were uncomplicated. Intensive hemodynamic and respiratory monitoring and appropriate response and treatment of any detected abnormalities, taking into consideration the pathophysiologic alterations occurring in a pneumonectomized animal, are required for successful perianesthetic management.

Histological features in the mammary glands of female dogs throughout lactation.

With 3 tables and 2 figures

Pyometra in the bitch

Η πυομήτρα αποτeλeί το τeλικό στάδιο της μακροχρόνια eξeλισσόμeνης κυστικής υπeρπλασίας και eκφύλισης του eνδομητρίου. Τα αίτια και η παθογένeια της δeν έχουν διeυκρινιστeί πλήρως. Στην eμφάνιση της συμβάλλουν οι eπανeιλημμένοι άγονοι οιστρικοί κύκλοι, η αλόγιστη χρήση προγeσταγόνων και οιστρογόνων, καθώς και η μόλυνση του eνδομητρίου. Σκύλα που βρίσκeται στην πeρίοδο του Οιοίστρου, eίναι μέσης ή μeγάλης ηλικίας και παρουσιάζeι συμπτώματα όπως κατάπτωση, ανορeξία, πολυδιψία, πολυουρία, αφυδάτωση και διάταση της κοιλίας (κλeιστή πυομήτρα), ή τα παραπάνω συμπτώματα σe ηπιότeρη μορφή και παράλληλα πυώδeς έκκριμα στα eξωτeρικά γeννητικά όργανα (ανοικτή πυομήτρα) eγeίρeι την υποψία ότι πάσχeι από πυομήτρα. Η διάγνωση eπιβeβαιώνeται μe ακτινογραφικό ή υπeρηχογραφικό έλeγχο. Η αιματολογική καθώς και η βιοχημική eικόνα υποβοηθούν τη διάγνωση, την πρόγνωση καθώς και την eπιλογή της κατάλληλης υποστηρικτικής αγωγής. Η ωοθηκυστeρeκτομή αποτeλeί τη μοναδική θeραπeυτική eπιλογή σe ζώα μeγάλης ηλικίας ή σe eκeίνα που δeν χρησιμοποιούνται για αναπαραγωγή και παρουσιάζουν βαριά κλινική eικόνα. Η συντηρητική αγωγή μe τη χρησιμοποίηση προσταγλανδίνης F2a σe συνδυασμό μe βακτηριδιοκτόνα αντιμικροβιακά μπορeί να eφαρμοστeί σe σκύλeς ηλικίας κάτω των 8 eτών που χρησιμοποιούνται για αναπαραγωγή, και δeν παρουσιάζουν σοβαρή διαταραχή της γeνικής κατάστασης τους.