Effect of cryopreservation on sperm DNA fragmentation and apoptosis rates in the testicular tissue of domestic cats.

The objectives of the present study were to evaluate the damage caused by cryopreservation on sperm DNA and estimate the percentage of cell apoptosis in tissue after thawing. Testicles of cats were sectioned into of 0.3 cm3 and 0.5 cm3 fragments and evaluated for DNA damage using acridine orange and semi-quantitatively through histo-morphological and immunohistochemical methods (caspase-3). Other fragments were placed in cryotubes with diluent containing either 3% glycerol or 3% propanediol, and were cryopreserved. Evaluation using acridine orange indicated there was a difference with use of propanediol and glycerol on DNA damage in 0.5 cm3fragments, with the latter being more effective than the former for cryopreservation. Results from histomorphological evaluations indicated there was a greater cell integrity among germ cells that were not cryopreserved, based on criteria assessed (detachment of cells from basal membrane, retraction of seminiferous tubule epithelium, visibility of the spermatogonia nucleoli and nuclear spermatogonia condensation), for both sizes of fragments. The values for these variables decreased after cryopreservation, with there being no differences as a result of size of fragment stored or between cryoprotectants used (P > 0.05). The staining for caspase-3 differed for the cytoplasm, nuclei and germ cells. Assessment of these staining patterns indicated the fresh fragments had an amount of cell damage and there was a similar amount of damage detected in cryopreserved fragments. This finding indicated that there was considerable efficacy in preserving the tissue fragments with use of the freezing protocols that were evaluated in this study.

An update on the cecectomy technique in roosters anesthetized with isoflurane used in subsequent amino acid digestibility experiments.

This work aims to study an alternative technique of cecectomy in roosters using inhalation anesthesia for subsequent use in digestibility experiments. A total of 30 adult chickens of Leghorn breed were used with an average age of 27 wk. The birds were preoxygenated, and the anesthetic induction was performed using isoflurane diluted in oxygen. After proper muscle relaxation, endotracheal intubation was performed using a Murphy catheter and kept in anesthesia under mechanical ventilation with a constant monitoring of electrocardiography variables, heart rate, oxyhemoglobin saturation, and body temperature during the surgical procedure. An incision of approximately 3 cm was made between keel and cloaca in order to expose and extirpate the cecum followed by a simple ligature. The opening of the peritoneal cavity was closed in 2 ways: Sultan suture technique was used for closing the abdominal wall and modified Cushing intradermic continuous points for closing the skin. The time for anesthesia induction and preoperational period noted to be 10 ± 2 min. Surgical procedures completed in 7 ± 1.5 min. No intraoperatively and postoperatively harm observed in animals. Water was immediately provided after the birds returned to cages and feed offered after 24 h of surgery. In remaining birds, no abnormalities were observed during and after the experimental period (3 mo). The present study describes a promising update on cecectomy technique regarding anesthesia induction and surgical procedures in roosters using potential drugs and safer surgical materials without any trans- and postoperative complications.

Ultrasonographic characteristics of accessory sex glands and spectral Doppler indices of the internal iliac arteries in peri- and post-pubertal Dorper rams raised in a subtropical climate.

The aim of the present study was to determine and compare ultrasonographic characteristics of accessory sex glands and spectral Doppler indices of the internal iliac arteries in peri- and post-pubertal rams raised in a subtropical climate. Forty-five Dorper rams were examined (24 rams aged 8-11 months and 21 rams aged 12-24 months). Digital images of all accessory sex glands were subjected to morphometric and echotextural analyses, the latter using commercially available image analytical software Image ProPlus®. Physical and morphological characteristics of semen and serum concentrations of testosterone were also determined. The dimensions of the prostate gland (12.9±1.2 compared with 14.2±2.7mm; mean±standard deviation) and bulbourethral glands (13.7±1.3 compared with 14.7±1.8mm) were greater (P=0.04) in sexually mature compared with peri-pubertal rams. Pixel intensity of vesicular (181.5±20.8 compared with 164.8±26.8, P=0.02) and bulbourethral gland parenchyma (166.9±16.9 compared with 141.8±29.1, P=0.001) was greater in peri-pubertal compared with sexually mature rams. Semen could be collected by ejaculation into the artificial vagina from 38% (8/21) of post-pubertal rams and 21% (5/24) of peri-pubertal animals (P=0.03). Semen volume was positively correlated with peak systolic velocity (PSV) and end-diastolic velocity (EDV) in the internal iliac arteries (r=0.79, P=0.001 and r=0.67, P=0.01, respectively), while spermatic vigor and progressive motility were inversely related to circulating concentrations of testosterone (r=-0.69, P=0.009 and r=-0.61, P=0.03, respectively). In summary, the attainment of sexual maturity in the rams of the present study was associated with an enlargement of the prostate and bulbourethral glands, and with the shift in echotextural attributes of vesicular and bulbourethral glands. Circulating testosterone concentrations and Doppler blood flow indices of the ram's internal iliac arteries are significant predictors of sperm progressive motility, vigor and the amount of ejaculate.

Evaluation of different fragment sizes and cryoprotectants for cryopreservation of feline testicular tissues.

This study aimed to evaluate tissue damage of feline testicles sectioned in two different sizes (0.3 or 0.5 cm3 ) and submitted to different cryoprotectants (propanediol or glycerol). Testicles obtained from 12 domestic cats were sectioned in 0.3 and 0.5 cm3 sized pieces and immediately evaluated by TBARS and semi-quantitatively by histomorphology. The remaining fragments were placed in cryotubes with 1 ml Egg yolk Tris Equex STM extender containing 3% glycerol or 3% propanediol and cryopreserved by fast-freezing technique. Frozen-thawed fragments were also evaluated by TBARS and histomorphology. Statistical analysis was performed using one-way ANOVA with Student-Newmann-Keuls post hoc test, with p < .05. Fresh and cryopreserved tissues generally exhibited a similar morphology concerning detachment of cells from the basement membrane and observation of nucleoli, with a great proportion scored as 0 (no alteration). When present, alterations were slight and the morphology was considered to be good (most classified in scores 1). Pyknosis was the main anomaly observed as score 2 in 54.6% and 58.4% of 0.3-cm3 fragments cryopreserved in propanediol and glycerol, respectively (16.7% scored 2 in fresh tissue). In TBARS evaluation, 0.5-cm3 fragments cryopreserved in glycerol produced less free radical compared to the 0.3 cm3 cryopreserved in glycerol or propanediol. Our results showed that glycerol was more efficient than propanediol to cryopreserve 0.5-cm3 fragments; this might be attributed to the fact that glycerol molecular weight is larger than propanediol and so its perfusion in the testicular tissue is slower.

Matrix-assisted laser desorption/ionization imaging mass spectrometry for the spatial location of feline oviductal proteins.

With the purpose of identifying factors involved in early stages of embryo development in the domestic cat, matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) was used for the first time to describe the spatial localization of proteins in the oviducts of queens. Oviducts were obtained from two 2 and 4 years old cross-bred queens, divided into three segments, snap-frozen in liquid nitrogen and then stored at -80°C until use. Next, they were sectioned in a cryostat, fixed on ITO (indium tin oxide) conductive glass slides for MALDI-IMS and serial sections were collected on microscope slides for histology. As confirmed by histology, MALDI-IMS was able to show contrasting protein distributions in the oviductal infundibulum, ampulla and isthmus. Mass spectra were characterized by abundant ions of m/z 1,259, 4,939, 4,960 and 10,626, which have been tentatively attributed to keratin, thymosin ß10, thymosin ß4 and S100, respectively. Keratin and thymosins are involved in the biological response to tissue damage. S100 proteins are calcium-modulated proteins implicated in a variety of cellular activities, including cell differentiation and regulation of cell motility. These results suggest that protein composition differs between segments of the cat oviduct, which corresponds to morphological changes within these sections. Further functional studies could elucidate the effects of these proteins on feline reproductive physiology.