Free gossypol supplementation frequency and reproductive toxicity in young bulls.

The aim of the present study was to analyze seminal quality of young bulls subjected to different frequencies of gossypol supplementation. Forty-eight Nellore bulls, with 19 months of age and weighing 357.8 ±â€¯7.2 kg, were used in this study. Animals were fed with 10.5 kg of standard supplement containing free-gossypol from whole cottonseed (WCS) at the following frequency: 3x/week (G3x), 5x/week (G5x) or 7x/week (G7x - Control). Additionally, a negative control was provided, and the treated animals received only mineral supplement (MM) ad libtum. The experiment lasted for 84 days and semen was collected at the beginning and at the end for analysis and cryopreservation. Fresh semen was used for initial analysis and plasma membrane integrity and sperm morphology were also determined. General motility using computer assisted sperm analysis (CASA), plasma and acrosomal membranes integrity, mitochondrial activity, and induced oxidative stress were assessed in post-thawed semen. The study design was completely randomized. Parametric data were analyzed by ANOVA and non-parametric data by the Wilcoxon test, using the statistical program SAS. Level of significance was set at 5%. Supplementation with WCS, regardless the frequency, increased total (P = .009) and head (P = .005) defects in comparison to animals receiving only forage and mineral supplement. Infrequent supplementation, particularly 5 times in the week (G5X), increased head (P = .026) and midpiece (P = .014) abnormalities. Sperm motility in fresh semen was lower in animals that received daily supplementation than those supplemented on alternate days (P = .021). Additionally, animals supplemented daily showed lower percentage of spermatozoa with intact acrosome compared to those supplemented on alternate days (P = .005). Thus, regardless the frequency of supplementation, free-gossypol supplementation affects sperm quality. Although the amount of free gossypol supplied weekly was the same among treatments, daily supplementation compromised sperm kinetics, differently from infrequent supplementation that led to sperm defects developed during spermatogenesis.

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.

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.

Laparoscopic ovum collection in sheep: gross and microscopic evaluation of the ovary and influence on ooctye production.

Gross and microscopic evaluations of the ovaries of sheep subjected to successive ovum collection were performed with the objective of identifying if there was interference in oocyte production and morphology of gonads. Gross evaluation of the internal genital tract was also performed. Eighteen ewes of the Santa Inês breed were randomly distributed into three experimental groups of six animals each; G0, G1 and G9 with no, one and nine repetitions, respectively. Estrous synchronization was achieved with a short protocol using MAP followed by single dose stimulation with 80mg of FSHp and 300 IU of eCG (IM). Laparoscopic ovum pick-up was performed 36h later, with 7-day intervals. The number of visualized follicles, aspirated follicles and oocytes recovery was recorded. After the last intervention, ovariectomy was performed for evaluation of gross and microscopical appearance and existence of lesions caused by follicular puncture, which were classified as absent (0), mild (1), moderate (2) and severe (3). The number of visualized follicles, aspirated follicles and oocytes recovery were 13.2±2.0, 11.3±3.0 and 5.8±2.3, respectively, with a recovery rate of 51.7%. No statistical difference was found between the nine sessions (P>0.05). Nine sessions of superovulation and ovum collection procedures did not cause ovarian lesions and did not interfere with the production of follicles in ewes of the Santa Inês breed.

Effects of carprofen administered by different routes to control experimental uveitis in dogs / Efeitos do carprofeno, aplicado por diferentes vias, no controle de uveítes experimentais em cães

Efficacy of carprofen, administered by different routes, was studied in experimental uveitis in dogs. Anterior chamber paracenteses was accomplished at two different moments (M0 and M1), with a five hour interval between them. At M0 and M1, 0.2mL of aqueous humor was collected and quantitation of total protein and prostaglandin E2 (PGE2) were determined. Four groups were formed (n=8), which received carprofen at the end of M0, by the following routes: subcutaneous (GIm), subconjunctival (GII), and topical (GIII). A fourth group that received no treatment was instituted (Control). Conjunctival histopathology of the GII animals was performed. In all groups, values of protein and PGE2 significantly enhanced at M1; however, they did not significantly change among groups at M1. Inflammatory exudate of acute character and mild hemorrhage were seen at histopathology after carprofen administration. Carprofen was unable to inhibit PGE2 synthesis and the protein influx to the anterior chamber by any of the tested routes. However, the reduction of 44 percent in protein levels (topical) suggests that the agent can be used by this route as an adjuvant to control uveitis in dogs.

Estudaram-se os efeitos do carprofeno, aplicado por diferentes vias, em uveítes experimentais em cães. Realizou-se paracentese de câmara anterior em dois momentos (M0 e M1), com intervalo de cinco horas entre si. Em M0 e M1, colheram-se 0,2mL de humor aquoso e determinaram-se as concentrações de proteína total e de prostaglandina E2 (PGE2). Constituíram-se quatro grupos (n = 8), que receberam carprofeno ao final de M0 pelas vias subcutânea (GI), subconjuntival (GII) e tópica (GIII). Um quarto grupo não recebeu tratamento (controle). Procedeu-se à avaliação histopatológica nos indivíduos do GII. Em todos os grupos, encontrou-se aumento significativo dos níveis proteicos e de PGE2 em M1. Não se observou diferença significativa, em M1, entre os grupos para nenhum dos parâmetros estudados. Exsudado inflamatório de caráter agudo e hemorragia discreta foram vistos à histopatologia após a aplicação do fármaco. O carprofeno foi ineficaz em inibir a síntese de PGE2 e o influxo de proteínas para a câmara anterior, por qualquer uma das vias testadas. Contudo, a redução de 44 por cento nos níveis de proteínas (via tópica), sugere que por esta via ele pode ser utilizado como adjuvante no controle da uveíte em cães.

Detecção ultrassonográfica do ovário fetal em loba-guará (Chrysocyon brachyurus): relato de caso / Ultrasonographic detection of fetal ovary in maned wolf (Chrysocyon brachyurus): case report

Identificaram-se, por ultrassonografia, os ovários fetais e o sexo dos fetos em uma loba-guará. Ao exame ultrassonográfico, foi possível identificar estruturas fetais e realizar medidas (relação entre diâmetro cranial e abdominal dos fetos) que indicavam idade fetal de 59 dias. Observaram-se: estruturas torácicas e abdominais formadas, diafragma, membros, estruturas do crânio definidas, coluna vertebral, medula, costelas, batimentos cardíacos normais, movimentação fetal, câmaras e valvas cardíacas, grandes vasos, rins, peristaltismo intestinal e ovários fetais. O exame ultrassonográfico foi eficaz nessa espécie, pois foi possível observar estruturas fetais para a avaliação da viabilidade fetal e, também, identificou-se a imagem ovariana em um dos fetos, mostrando-se importante para estudos de sexagem fetal.

The ovaries and the gender of the fetuses in a female maned wolf (Chrysocyon brachyurus) were identified by ultrasound examination. It was possible to identify fetal structures and to determine measures, i.e., cranial and abdominal diameters and these elements indicated 59-day of fetal age. The main visualized structures were diaphragm, members, cranium, spine, marrow, ribs, cardiac chambers and valves, veins and arteries, kidneys, and fetal ovaries. Normal heartbeats and fetal and bowel movements were recorded. The ultrasound examination was an effective method in this species, since enabled the correct observation of the fetal structures as well as viability; in addition, proved to be an important method for fetal sexing determination.

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.