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.

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.

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.

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 with DAP 213 and cryotop of ex situ and in situ feline cumulus-oocyte complexes.

This study was undertaken to compare cryotolerance, in terms of viability and resumption of meiosis after warming and culture (24 and 48 h), of ex situ (isolated) and in situ (enclosed in the ovarian tissue) feline cumulus-oocyte complexes (COCs) vitrified with DAP 213 (2 m DMSO, 1 m acetamide, 3 m propylene glycol) in cryotubes or Cryotop method. Ovaries were harvested from 49 pubertal queens. Of each pair of ovaries, one was dissected to release COCs randomly divided into three groups: fresh COCs (control), ex situ COCs vitrified with DAP 213 and Cryotop. The cortex of the other ovary was sectioned into small fragments (approximately 1.5 mm(3)) and randomly assigned to be vitrified by DAP 213 or Cryotop. After warming, ex situ and in situ (retrieved form vitrified ovarian tissue) COCs were matured in vitro. Viability of oocytes was highly preserved after warming and culture in all treatments. Proportions of oocytes surrounded by complete layers of viable cumulus cells were remarkably decreased (p < 0.00001) in both vitrification procedures compared to fresh oocytes. Resumption of meiosis occurred in all treatments. After 24 h of culture, results were similar in ex situ and in situ vitrified oocytes regardless of the vitrification protocol used (range 29-40%), albeit lower (p < 0.05) than those of fresh oocytes (65.8%). After 48 h of culture, ex situ oocytes vitrified with Cryotop achieved the rates of meiosis resumption similar to fresh oocytes (53.8% vs 67.5%; p > 0.05) and ex situ and in situ oocytes vitrified with DAP 213 showed similar rates of resumption of meiosis. These findings demonstrated that DAP 213 and Cryotop preserve the viability of ex situ and in situ oocytes, but cumulus cells are highly susceptible to vitrification. However, the capability to resume meiosis evidences that feline immature oocytes vitrified as isolated or enclosed in the ovarian cortex have comparable cryotolerance.

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.

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.

Characterization of polymorphisms in the mannose-binding lectin gene promoter among human immunodeficiency virus 1 infected subjects.

The present study investigated the prevalence of mutations in the -550 (H/L) and -221 (X/Y) mannose-binding lectin (MBL) gene promoter regions and their impact on infection by human immunodeficiency virus 1 (HIV-1) in a population of 128 HIV-1 seropositive and 97 seronegative patients. The allele identification was performed through the sequence-specific primer polymerase chain reaction method, using primer sequences specific to each polymorphism. The evolution of the infection was evaluated through CD4+ T-lymphocyte counts and plasma viral load. The allele and haplotype frequencies among HIV-1-infected patients and seronegative healthy control patients did not show significant differences. CD4+ T-lymphocyte counts showed lower levels among seropositive patients carrying haplotypes LY, LX and HX, as compared to those carrying the HY haplotype. Mean plasma viral load was higher among seropositive patients with haplotypes LY, LX and HX than among those carrying the HY haplotype. When promoter and exon 1 mutations were matched, it was possible to identify a significantly higher viral load among HIV-1 infected individuals carrying haplotypes correlated to low serum levels of MBL. The current study shows that haplotypes related to medium and low MBL serum levels might directly influence the evolution of viral progression in patients. Therefore, it is suggested that the identification of haplotypes within the promoter region of the MBL gene among HIV-1 infected persons should be further evaluated as a prognostic tool for AIDS progression.

Role of adenosine on intestinal ischemia-reperfusion injury in rabbits.

To study if the treatment with adenosine (ADO), an agonist of adenosine receptors, attenuates intestinal dysfunction caused by ischemia (I) and reperfusion (R), we treated rabbits with ADO (15 mg x kg(-1), intravenously) or saline solution (SS) to I (60 minutes) before occlusion of superior mesenteric artery and/or R (120 min). After I or I/R, isolated jejunal segments (2 cm) were mounted in an organ bath to study nerve-mediated contractions stimulated by electrical pulses or KCl using a digital recording system. Thin jejunal slices were stained (hematoxylin and eosin) for analysis by optical microscopy. Compared to the sham group, the jejunal contractions were similar in I + ADO, but reduced in I + SS, I/R + SS, and I/R + ADO groups. We concluded that the jejunal enteric nerves were damaged in I + SS, I/R + SS, and I/R + ADO, but not in I + ADO group. These results suggested that ADO attenuated intestinal dysfunction due to I, but not to R.

Polymorphism in the promoter region of the mannose-binding lectin gene among human T-cell lymphotropic virus infected subjects.

The present study investigated the frequency of the mutations at positions -550 and -221 of the mannose-binding lectin (MBL) gene in a sample of 75 human T-cell lymphotropic virus (HTLV) infected patients and 96 HTLV seronegative controls, in order to evaluate the occurrence of a possible association between the polymorphism and HTLV infection. A sequence specific primer-polymerase chain reaction was used for discrimination of the polymorphism. The analysis of allele frequencies at position -550 did not show any significant differences between HTLV infected group and controls, but there was a significant difference at position -221. The comparative analysis of haplotypes frequencies were not significant, but the genotype frequencies between the two groups, revealed a higher prevalence of genotype LYLX (25.3%), associated with medium and low MBL serum levels among HTLV infected subjects. The odds ratio estimation demonstrated that the presence of genotype LYLX was associated with an increased risk of HTLV infection (p = 0.0096; 1.38 < or = IC95% < or = 7.7605). There was no association between proviral load and the promoter polymorphism, but when promoter and exon 1 mutations were matched, it was possible to identify a significant higher proviral load among HTLV infected individuals carrying haplotypes correlated to low serum levels of MBL. The present study shows that the polymorphism in the promoter region of the MBL gene may be a genetic marker associated with HTLV infection, and emphasizes the need for further studies to determinate if the present polymorphism have any impact on diseases linked to HTLV infection.

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.

Polymorphism in the promoter region of the mannose-binding lectin gene among human T-cell lymphotropic virus infected subjects

The present study investigated the frequency of the mutations at positions -550 and -221 of the mannose-binding lectin (MBL) gene in a sample of 75 human T-cell lymphotropic virus (HTLV) infected patients and 96 HTLV seronegative controls, in order to evaluate the occurrence of a possible association between the polymorphism and HTLV infection. A sequence specific primer-polymerase chain reaction was used for discrimination of the polymorphism. The analysis of allele frequencies at position -550 did not show any significant differences between HTLV infected group and controls, but there was a significant difference at position -221. The comparative analysis of haplotypes frequencies were not significant, but the genotype frequencies between the two groups, revealed a higher prevalence of genotype LYLX (25.3 percent), associated with medium and low MBL serum levels among HTLV infected subjects. The odds ratio estimation demonstrated that the presence of genotype LYLX was associated with an increased risk of HTLV infection (p = 0.0096; 1.38 < IC95 percent < 7.7605). There was no association between proviral load and the promoter polymorphism, but when promoter and exon 1 mutations were matched, it was possible to identify a significant higher proviral load among HTLV infected individuals carrying haplotypes correlated to low serum levels of MBL. The present study shows that the polymorphism in the promoter region of the MBL gene may be a genetic marker associated with HTLV infection, and emphasizes the need for further studies to determinate if the present polymorphism have any impact on diseases linked to HTLV infection.