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.

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.

Viability and growth of feline preantral follicles in vitro cultured with insulin growth factor and epidermal growth factor supplemented medium.

In vitro culture of ovarian preantral follicles has emerged as a reproductive technology aimed at obtaining large amount of oocytes for in vitro embryo production. The addition of growth factors (GF) in the in vitro culture of preantral follicles of different species has provided superior results of follicular development, antrum formation and proliferation of granulosa cells. However, there are only few reports regarding the use of these factors on feline preantral follicle in vitro culture. Thus, the aim of this study was to investigate the effect of a combination of IGF-1 and EGF on in vitro viability and growth of preantral follicles and enclosed oocytes collected from domestic cats. A total of 64 follicles characterized by multilayer granulosa cells were isolated and individually cultured for 6 days (T6) in minimum essential medium supplemented with IGF-1+ EGF (100 ng/ml each) or without (control). A higher percentage of follicles were viable after culture with GF than without, and an increase in size when IGF-1+ EGF were added to the medium (170 ± 32.4 µm (T0) vs. 201 ± 22.3 µm (T6); p < .05) was observed. An increase in the diameter was also observed in follicles cultured without GF, but this increase was only 8.3% compared to 15.4% of those cultured with GF (p < .05). No differences were found in the diameter of oocytes contained in follicles cultured in the non-supplemented or supplemented media (107.9 ± 11.8 µm (T0) vs. 113.2 ± 15.6 µm (T6); p > .05). These data suggest that the addition of IGF-1 and EGF to the culture medium promotes the in vitro development of preantral follicles of cats.

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.

Changes in acetylation of lysine 5 on histone H4 in canine oocytes following in vitro maturation.

Post-translational modifications of histones, such as acetylation, are involved in regulating chromatin remodelling and gene expression. Proper in vitro maturation (IVM) of canine oocytes, for many reasons, is up to now inefficient. This study aimed to evaluate the post-translational histone H4 acetylation at lysine 5 (H4K5) in immature and post-IVM canine oocytes. Oocyte nuclear stage was assessed using Hoechst 33342 staining. Acetylation patterns were determined by indirect immunofluorescence staining of immature and post-IVM oocytes, using an antibody against the acetylated lysine 5 residue on histone 4 (H4K5ac). The experiment was repeated four times, with a total of 7-17 oocytes evaluated per stage. Immunofluorescence signal was quantified using the NIHimagej software. Data were expressed as a percentage of the average fluorescence intensity of the specific antibody over the intensity of DNA, as determined by Hoescht staining. H4K5ac displayed a significantly higher acetylated pattern in immature oocytes (0.97 ± 0.08) when compared to post-IVM oocytes at different nuclear stages. There was a decrease in the fluorescence level of the matured oocytes with the progression of meiosis (GVBD: 0.47 ± 0.06 and MI/MII: 0.35 ± 0.04). Similarly to other domestic species, we hypothesized that post-translational modification of histone acetylation takes place during meiosis of in vitro matured canine oocytes. However, it remains to be investigated whether these changes occur during in vivo maturation.

In vitro maturation of oocytes from Santa Ines ewes subjected to consecutive sessions of follicular aspiration by laparoscopy.

The success of embryo production in vitro depends upon the use of an efficient oocyte retrieval technique, and the best results have been obtained by laparoscopic aspiration. The aim of this study was to evaluate the effect of consecutive sessions of follicular aspiration on the quantity, quality and in vitro maturation competence of oocytes obtained from ewes subjected to hormonal stimulation. Six Santa Ines ewes underwent nine sessions of follicular aspiration by laparoscopy with a 7-day interval between sessions, totalling 56 aspirations. After 24 h of culture, oocytes were stained and classified according to the stage of nuclear and cytoplasmic maturation. Oocyte retrieval rate was 61.4 ± 2%, resulting in a total of 249 oocytes. No significant variation was observed between sessions (p > 0.05). The average number of oocytes retrieved from each ewe was 6.4 ± 2 per session and 42 ± 4 in total. No significant difference was observed between the frequencies of the different stages of nuclear maturation: 32.72% mature, 40.74% immature and 26.54% degenerated/indeterminate oocytes; however, a significant difference was observed between the frequencies of the different stages of cytoplasmic maturation: 10.7% mature, 73.25% immature and 16.05% degenerated/indeterminate oocytes. No significant difference was observed in nuclear or cytoplasmic maturation between the weeks of procedure. We conclude that after nine consecutive sessions of follicular aspiration, the quantity and quality of retrieved oocytes remained unchanged as well as the levels of nuclear and cytoplasmic maturation obtained, demonstrating the viability of this technique for repetitive follicular aspirations on the same donor.

Vitrification of immature feline oocytes with a commercial kit for bovine embryo vitrification.

The aim of this study was to evaluate the suitability of a commercial kit for bovine embryo vitrification for cryopreserving cat oocytes and to evaluate comparatively the effects of its use with slow freezing procedure on cryotolerance in terms of morphology and oocyte resumption of meiosis. Germinal vesicle stage oocytes isolated from cat ovaries were either vitrified (n = 72) using a vitrification kit for bovine embryo or slow frozen (n = 69) by exposing oocyte to ethylene glycol solution before being transferred to a programmable embryo freezer. After thawing and warming, oocytes were cultured for 48 h and then were examined for meiosis resumption using bisbenzimide fluorescent staining (Hoechst 33342). Fresh immature oocytes (n = 92) were used as the control group. The proportion of oocytes recovered in a morphologically normal state after thawing/warming was significantly higher in frozen oocytes (94.5%) than in the vitrified ones (75%, p < 0.01). Morphological integrity after culture was similar in vitrified (73.6%) and slow frozen oocytes (76.8%); however, only 37.5% of the morphologically normal oocytes resumed meiosis after vitrification compared to 60.9% of those submitted to slow freezing procedure (p < 0.01). Fresh oocytes showed higher morphological integrity (91.3%) and meiosis resumption rates (82.6%, p < 0.002) than cryopreserved oocytes, irrespective of the procedure used. These results suggest that immature cat oocytes vitrified with a kit for bovine embryos retain their capacity to resume meiosis after warming and culture, albeit at lower rates than slow frozen oocytes. Vitrification and slow freezing methods show similar proportions of oocytes with normal morphology after culture, which demonstrate that thawed and warmed oocytes that resist to cryodamage have the same chances to maintain their integrity after 48 h of culture.

Effect of vitrification of feline ovarian cortex on follicular and oocyte quality and competence.

Cryopreservation of ovarian cortex has important implications in the preservation of fertility and biodiversity in animal species. Slow freezing of cat ovarian tissue resulted in the preservation of follicular morphology and in the follicular development after xenografting. Vitrification has been recently applied to ovarian tissues of different species, but no information is available on the effect of this method on feline ovarian cortex. Moreover, meiotic competence of fully grown oocytes isolated from cryopreserved tissue has not been reported. The aim of this study was to evaluate the effect of vitrification of feline ovarian cortex on follicular morphology and oocyte integrity, as well as meiotic competence. A total of 352 fragments (1.5-2 mm(3) ) were obtained from ovarian cortical tissues: 176 were vitrified and 176 were used fresh as control. Histological evaluation of fresh and vitrified fragments showed intact follicles after cryopreservation procedures with no statistically significant destructive effect from primordial to antral follicles. After IVM, oocytes collected from vitrified ovarian fragment showed a higher proportion of gametes arrested at germinal vesicle (GV) stage compared to those isolated from fresh control tissue (33.8% vs 2.9%; p < 0.001). However, oocytes isolated from vitrified tissues were able to resume meiosis, albeit at lower rate than those collected from fresh tissues (39.8% vs 85.9%; p < 0.00001). Vitrification induced changes in the organization of cytoskeletal elements (actin microfilaments and microtubules) of oocytes, but significantly only for actin network (p < 0.001). Finally, chromatin configuration within the GV was not affected by the cryopreservation procedure. Our study demonstrated that vitrification preserves the integrity of ovarian follicles and that oocytes retrieved from cryopreserved tissue maintain the capability of resuming meiosis. To our knowledge, this has not previously been reported in the cat.

In vitro survival of follicles collected from domestic cats' ovaries at different stages of oestrous cycle and cultured with IGF-1.

Optimal conditions for in vitro culture of feline ovarian follicles have not yet been defined. Follicular development is regulated by intraovarian growth factors, as insulin-like growth factor (IGF-1), and during the different stages of the oestrous cycle, follicles are exposed to specific hormonal environments. The aim of this study was to investigate the effect of IGF-1 on in vitro growth and granulosa cell (GC) viability of preantral follicles collected from domestic cats at follicular and luteal phases of the oestrous cycle. Oestrus and ovulation were induced in 12 cats. A total of 39 and 32 follicles collected at the follicular and luteal phases, respectively, were individually cultured in vitro for 6 days in minimum essential medium media supplemented with or without IGF-1 (100 ng/ml). Follicles collected during the follicular phase and cultured without IGF-1 displayed a significant increase in size and higher GC viability (46.5 ± 22.1 µm, 66.7%, respectively) than that of follicles collected at the luteal phase and cultured without IGF-1 (26.7 ± 14.4 µm, 50%, respectively; p < 0.05). In contrast, when IGF-1 was added to the culture medium, no differences were observed in size or GC viability between follicles collected at the two phases of the cycle. Nonetheless, follicles collected at the luteal phase and cultured with IGF-1 had a significant increase in their diameter and GC viability (31.9 ± 15.9 µm, 63.6%, respectively) than that cultured without IGF-1 (26.7 ± 14.4 µm, 50%, respectively; p < 0.05). These data suggest that in vitro growth and GC survival of feline preantral follicles are affected by the oestrous cycle phase, and the IGF-1 exerts a positive effect on follicles collected at the luteal phase.

Chemical composition of lipids present in cat and dog oocyte by matrix-assisted desorption ionization mass spectrometry (MALDI- MS).

The aim of the present study was to investigate the level of information on the chemical structures and relative abundances of lipids present in cat and dog oocytes by matrix-assisted laser desorption mass spectrometry (MALDI-MS). The MALDI-MS approach requires a simple analysis workflow (no lipid extraction) and few samples (two or three oocytes per analysis in this work) providing concomitant profiles of both intact phospholipids such as sphingomyelins (SM) and phosphatidylcholines (PC) as well as triacylglycerols (TAG). The lipids were detected in oocytes by MALDI using dihydroxybenzoic acid (DHB) as the matrix. The most abundant lipid present in the MS profiles of bitch and queen oocytes was a PC containing 34 carbons and one unsaturation [PC (34:1)]. Oocytes of these two species are characterized by differences in PC and TAG profiles detected qualitatively as well as by means of principal component analysis (PCA). Cat oocytes were mainly discriminated by more intense C52 and C54 TAG species and a higher number of unsaturations, indicating predominantly linoleic and oleic fatty acyl residues. Comparison of the lipid profile of bitch and queen oocytes with that of bovine oocytes revealed some similarities and also some species specificity: TAG species present in bovine oocytes were also present in bitches and queens; however, a more pronounced contribution of palmitic, stearic and oleic fatty acid residues was noticed in the lipid profile of bovine oocytes. MALDI-MS provides novel information on chemical lipid composition in canine and feline oocytes, offering a suitable tool to concomitantly monitor, in a nearly direct and simple fashion the composition of phospholipids and TAG. This detailed information is highly needed to the development of improved protocols for in vitro culture and cryopreservation of cat and dog oocytes.

Effects of hormonal supplementation on nuclear maturation and cortical granules distribution of canine oocytes during various reproductive stages.

The aim of this study was to evaluate the effects of hCG, progesterone and oestradiol supplementation on nuclear and cytoplasmic maturation of canine oocytes cultured for 24, 48, 72 and 96 h. Oocytes obtained from 18 healthy bitches were divided into three groups according to their reproductive status (follicular, luteal and anoestrus stages) and cultured in TCM 199 + 25 UI/ml of hCG + 1 µg/ml of progesterone + 1 µg/ml of 17-ß oestradiol or without hormonal supplementation (control) for different periods. Then, they were stained with FITC-LCA-Hoescht for chromatin configuration and cortical granules distribution and evaluated under an epifluorescence microscope. Culture time and the influence of different stages of the oestrous cycle were also evaluated. The present study demonstrated that there was no significant difference among the reproductive stages. With regards to culture medium, only oocytes from the supplemented medium were able to complete meiosis; however, significant difference was only noticed in the percentage of MI stage oocytes (p < 0.05) in the follicular and luteal group at 72 h of culture. Most oocytes in germinal vesicle, germinal vesicle breakdown and metaphase I stage had cortical granules distributed throughout the cytoplasm (immature pattern), irrespective of the culture period (p < 0.05). Cortical granules distributed immediately beneath the plasma membrane (mature) was only observed in metaphase II stage oocytes, but not all of them presented matured cytoplasm. Our results reveal that cortical granules distribution in canine oocytes matured in vitro did not progressed in correspondence with nuclear stage changes and are in accordance with those from other species.

Leucogram and serum acute phase protein concentrations in queens submitted to conventional or videolaparoscopic ovariectomy / Leucograma e concentrações séricas de proteínas de fase aguda de gatas submetidas à ovariectomia convencional ou por videolaparoscopia

Thirty health queens were submitted to ovariectomy by conventional technique or by videolaparoscopy. In order to study the intensity of inflammatory response by means of acute phase protein analysis and white blood cell count, serum samples were taken before and until 144 hours after the surgical procedures. The protein concentrations that were significantly increased 24 hours after surgical procedures were: ceruloplasmin, hemopexin, haptoglobin, and α1-acid glycoprotein, 69.8 percent, 103.5 percent, 117.3 percent, and 199.0 percent, respectively, for conventional ovariectomy; and 22.3 percent, 46.1 percent, 79.8 percent, and 74.6 percent, respectively, for laparoscopic ovariectomy. Therefore, inflammatory response was more intense in queens submitted to conventional ovariectomy. Results indicate that the increase or decrease in acute phase proteins, as well as in white blood cells count, may be useful in the evaluation of inflammatory response induced by these surgical procedures.

Trinta gatas, saudáveis, foram submetidas à ovariectomia pela técnica convencional e por videolaparoscopia. Amostras de sangue foram obtidas com o objetivo de verificar a intensidade da resposta inflamatória por meio da análise das concentrações de proteinas de fase aguda e contagem de leucócitos antes e até 144 horas após procedimento cirúrgico. As proteínas que apresentaram aumento significativo 24 horas após a cirurgia foram: ceruloplasmina, hemopexina, haptoglobina e α1-glicoproteína ácida, 69,8 por cento, 103,5 por cento, 117,3 por cento e 199,0 por cento, respectivamente, para ovariectomia convencional, e 22,3 por cento, 46,1 por cento, 79,8 por cento e 74,6 por cento, respectivamente, para ovariectomia por videolaparoscopia. A resposta inflamatória foi mais evidente nas gatas submetidas à ovariectomia convencional. Os resultados mostram aumento e diminuição na concentração de proteínas de fase aguda e na contagem de leucócitos, podendo ser utilizados na avaliação da resposta inflamatória induzida por procedimentos cirúrgicos.

Age-related ultrasonography, cytology, and microbiologic exam of canine prostate / Ultrassonografia, citologia e exame microbiológico da próstata de cães em diferentes idades

The physiological parameters that could be reference for trustful diagnosis and prognosis of prostate disorders in dogs were obtained. Thirty six intact male dogs without clinical signs of neither prostatic nor reproductive disorders were allocated according the age in three groups. These animals were submitted to semen manual collection for microbiological exams; transabdominal ultrasonography to evaluate dimensions, ecogenicity, and texture of prostatic parenchyma and aspirative puncture with fine needle for cytological and microbiological analyses. Ultrasonography revealed that the predominant prostatic shape was round with regular surface. Dimensions varied according to age, being small in young animals and large in old ones. There was a positive correlation between prostatic dimensions and body weight. Microbiological exams detected microorganisms on seminal plasma from 11 dogs and prostate tissue aspirated from 10 animals, although they were healthy. Cytology did not reveal any inflammatory, proliferative, or neoplasic alteration in young and middle age dogs, but in three older dogs signs of hyperplasia/hypertrophy was found. It was observed positive correlation between age and cellular area but a negative correlation was observed between nucleus:cytoplasm ratio and craniocaudal dimension.

Obtiveram-se parâmetros fisiológicos que pudessem ser utilizados como referência para diagnóstico e prognóstico confiáveis de doença prostática em cães. Trinta e seis cães, sem sinais clínicos de doença prostática ou distúrbios reprodutivos, foram distribuídos em três grupos de acordo com a idade.Os animais foram submetidos à colheita manual de sêmen para exames microbiológicos, à ultrassonografia transabdominal, para avaliar as dimensões, a ecogenicidade e a ecotextura prostática, e à punção aspirativa com agulha fina, para análise citológica e microbiológica. A ultrassonografia revelou que a forma predominante da próstata foi globosa, com superfície de contorno regular. As dimensões variaram de acordo com a idade, sendo pequena em animais jovens e grande nos animais idosos. Houve correlação positiva entre as dimensões prostáticas e o peso corporal. Os exames microbiológicos detectaram microrganismos no plasma seminal de 11 cães e no tecido prostático aspirado de 10 animais, embora eles fossem saudáveis. A citologia não revelou nenhuma alteração inflamatória, proliferativa ou neoplásica nos cães jovens e de meia idade, mas, em três cães idosos foram encontrados sinais de hiperplasia/hipertrofia. Foi observada correlação positiva entre a idade e a área celular e correlação negativa entre a relação núcleo:citoplasma e a dimensão craniocaudal.

Evaluation of creatine kinase (CK) and aspartate aminotransferase (AST) activities after laparoscopic or conventional ovariectomy in queens.

Creatine kinase (CK) and aspartate aminotransferase (AST) are mainly muscle-specific enzymes, which can be associated with muscle tissue damage. The aim of this study was to assess the activities of CK and AST during the postoperative period, after conventional (G1) and videolaparoscopic ovariectomy (G2), in queens. A further group (G3) was subjected to anaesthesia only. Results demonstrate that there were significant differences between groups. The highest levels of CK were recorded in G1, however at a confidence level of p<0.05 there was no significant difference between groups during the first 6 hours after surgery. A significant (p<0.05) increase of CK values was identified between 0 h and 3 h in both groups (G1 and G2). Regarding AST activity there was no significant variation between groups, but again there was a significant difference between values at 0 h and 3h after surgery. In conclusion, ovariectomy performed by videolaparoscopy seems to cause less muscle damage when compared to the conventional method.

Intraabdominal torsion of a neoplastic testicle and prostatic cyst in a cryptorchid dog.

The present report describes a case of a nine-year old male bilaterally cryptorchid boxer presented with testicular torsion and concurrent prostatic cyst. Clinical signs included anorexia, locomotor difficulty and apathy. Abdominal palpation revealed the presence of a hard and painful mass in caudal abdomen. Ultrasonographic findings were compatible with testicular torsion and prostatic cyst, confirmed at surgery. Bilateral orchiectomy and omentalisation were performed. Histopathological examination of the torsed testicle revealed alterations consistent with seminoma.

Acquired vesicovaginal fistula secondary to ovariohysterectomy in a bitch: a case report

Uma cadela de sete anos de idade foi trazida ao hospital veterinário "Governador Laudo Natel" pois apresentava estrangúria há dois meses. Os sinais clínicos desenvolveram-se dois dias após ovário-histerectomia de eleição. Exames radiográficos e ultrasonográficos sugeriram piometra de coto ou granuloma cervical e fistula vesicovaginal. Duas laparotomias foram realizadas para desfazer as adesões, mas não houve melhora nos sinais clínicos observados. Iniciou-se tratamento médico e oito meses depois o animal, novamente avaliado, apresentava-se sadio porém ainda com sinais de estrangúria.