Use of platelet-rich plasma on in vitro maturation during bovine embryo production.

One of the crucial aspects to be considered for successful in vitro production (IVP) of embryos is the composition of the various media used throughout the stages of this reproductive biotechnology. The cell culture media employed should fulfill the metabolic requirements of both gametes during oocyte maturation and sperm development, as well as the embryo during its initial cell divisions. Most IVP protocols incorporate blood serum into the media composition as a source of hormones, proteins, growth factors, and nutrients. Numerous studies have suggested Platelet-Rich Plasma (PRP) as a substitute for fetal sera in cell culture, particularly for stem cells. Therefore, the objective of this study is to assess the potential use of PRP as a replacement for fetal bovine serum (FBS) during oocyte maturation for in vitro production of bovine embryos. During in vitro maturation (IVM), cumulus-oocyte complexes (COCs) were allocated into the following experimental groups: Group G1 (IVM medium with 5% PRP); Group G2 (MIV medium with 5% PRP and 5% SFB); Group G3 (MIV medium with 5% SFB); and Group G4 (MIV medium without either PRP or SFB). Subsequently, the cumulus-oocyte complexes were fertilized with semen from a single bull, and the resulting zygotes were cultured for seven days. Cleavage and blastocyst formation rates were assessed on days 2 and 7 of embryonic development, respectively. The quality of matured COCs was also evaluated by analyzing the gene expression of HSP70, an important protein associated with cellular stress. The results demonstrated that there were no significant differences among the experimental groups in terms of embryo production rates, both in the initial cleavage stages and blastocyst formation (except for the G4 group, which exhibited a lower blastocyst formation rate on D7, as expected). This indicates that PRP could be a cost-effective alternative to SFB in the IVP of embryos.

Do the blood anti-Müllerian hormone concentrations in young females correlate with the ovarian follicular population later in life in water buffalo?

In this longitudinal study, the anti-Müllerian hormone (AMH) levels in blood were determined in 32 Murrah buffalo females at 8, 10, 12, 16 and 19 months of age when females were synchronized and the antral follicular population (AFP) was estimated. Correlations of AFP to the AMH level at 19 months of age and retrospectively to younger ages were investigated. Then females were split into high and low AFP, and their AMH levels were compared for all ages and tested as predictors of AFP categories. The highest AMH level (p < .05) was detected at 8 months, reducing but not differing (p > .05) at 10, 12 and 16 months then reducing again (p < .05) at 19 months of age. The mean AFP was 17.6 ± 6.3 follicles, and it was positively correlated with AMH in all ages tested. High AFP females had approximately two times more antral follicles than low AFP (p < .05) and their AMH levels were higher (p < .01) than in low AFP ones in all ages. Only at 8 months, AMH levels can be used to precociously detect high AFP heifers (a cut-off point of 464.7 pg/mL; p < .05), while low AFP heifers could be detected by AMH measurements at 8, 10, 12 and 16 months of age (p < .05). We conclude that AMH of buffalo calves correlates with AFP of heifers later in life and depending on the age, its levels could be used to identify future females with low or high AFP.

Fertilization with follicular fluid reduces HSP70 and BAX expression on bovine in vitro embryos.

The in vivo fertilization process occurs in the presence of follicular fluid (FF). The aim of this study was to evaluate the effect of in vitro fertilization medium supplementation with 5% or 10% bovine follicular fluid (BFF) on the production of in vitro bovine embryos. FF was collected from ovarian follicles with a diameter of 8-10 mm, and cumulus-oocyte complexes (COCs) were co-incubated with sperm for 24 h in the commercial medium BotuFIV® (BotuPharma©), being distributed among the experimental groups: oocytes fertilized in a control medium; oocytes fertilized in a medium supplemented with 5% BFF; and oocytes fertilized in a medium supplemented with 10% BFF. After fertilization, the zygotes were cultured in vitro for 8 days. Embryo development was assessed through cleavage rates (day 2) and blastocyst formation rates (day 8). The relative expression of the genes OCT4, IFNT2, BAX, HSP70 and SOD2 was measured using the real-time polymerase chain reaction method. There was no difference (p > .05) among the different experimental groups in terms of cleavage rates and blastocyst formation rates. Regarding the gene expression results, only the blastocysts from oocytes fertilized with 10% BFF showed significantly lower expression of IFNT2 (p = .003) and SOD2 (p = .01) genes compared to blastocysts from oocytes fertilized in control medium alone, while there was no difference between blastocyst from oocytes fertilized in control medium and the ones from oocytes fertilized with 5% BFF. In addition to this, the blastocysts from oocytes fertilized with 5% BFF showed significantly reduced levels of expression of the heat shock protein HSP70 (p < .001) and the pro-apoptotic protein BAX (p = .015) compared to blastocysts from oocytes fertilized with control medium. This may indicate that lower supplementation of BFF to the IVF medium creates a more suitable environment for fertilization and is less stressful for the zygote.

Artesunate does not affect oocyte maturation and early embryo development of bovine.

Despite the cytotoxicity and embryotoxicity previously reported artesunate is a recommended drug to treat malaria for adults, children, and women in the first trimester of pregnancy. To address the putative effects of artesunate on female fertility and preimplantation embryo development, when the pregnancy is not detectable yet, artesunate was added to the oocyte in vitro maturation and in vitro embryo development of bovine. Briefly, in experiment 1 the cumulus-oocyte complexes (COCs) were in vitro matured for 18 h with 0.5, 1, or 2 µg/mL of artesunate or not (negative control) and then checked for nuclear maturation and subsequent embryo development. In experiment 2, the COCs were in vitro matured and fertilized without artesunate, which was added (0.5, 1, or 2 µg/mL) from the 1st to the 7th day of embryo culture along with a negative and a positive control group with doxorubicin. As a result, the use of artesunate on oocyte in vitro maturation did not differ from the negative control (p > 0.05) regarding nuclear maturation, cleavage, and blastocyst formation. Also, artesunate on in vitro embryo culture did not differ from negative control (p > 0.05) regarding cleavage and blastocyst formation, except for positive control, with doxorubicin (p < 0.05). In conclusion, under the conditions investigated, there was no evidence of artesunate toxicity on oocyte competence and the preimplantation period of in vitro embryo development in the bovine model, however, artesunate use still should be taken carefully as the outcome of implantation after oocytes and blastocysts exposure to artesunate remains unknown.

Artesunate added to in vitro maturation did not impair oocyte competence in bovine.Artesunate added to in vitro culture did not affect cleavage and blastocyst formation.No evidence of artesunate toxicity in oocytes and embryos of bovine was found.

Scrotal thermoregulation and sequential sperm abnormalities in buffalo bulls (Bubalus bubalis) under short-term heat stress.

Heat stress reduces the reproductive capacity of bulls raised in tropical climate. However, the reestablishment of scrotal thermoregulation and the dynamics of sperm defects emergence after stress are not completely known in buffaloes. Thus, the study aimed to evaluate the effect of short-term heat stress over scrotal thermoregulation and sperm attributes, relating them to spermatogenesis stages. Five buffalo bulls went through scrotal insulation during 48 h (from day 0 to day 2). Semen samples were collected every 7 days (from day -7 to day 49) and analyzed about the progressive motility, viability, and sperm morphology. Heat stress significantly destabilized scrotal thermoregulation (P < 0.001). Scrotal temperature was from 4.2 to 6.3 °C lower than the core body temperature, except on insulation days (P < 0.001), and returned to the basal condition five days after the removal of the stressing stimulus. More significant deleterious effects were observed in sperm morphology than in cell concentration, motility, and viability. The chronology of morphologic defects expression demonstrated tail defects (days 7-14), cytoplasmic droplets (days 14-28), and head defects (day 28), returning to pre-insulation condition 35 days after the thermal challenge. Thus, hyperthermia harmed more intensely spermatozoa in epididymal transit, elongated spermatids, and secondary spermatocytes. It is concluded that water buffalo bulls present a peculiar manifestation of sperm morphology after short-term stress, indicating an important difference related to the bovine species. Therefore, during the andrological evaluation of buffalo bulls, it is necessary to avoid the allometric extrapolation between these species.

Sperm chromatin protamination influences embryo development in unsexed and sexed bull semen.

Sex selection through sperm sorting offers advantages in regards selection pressure in high-producing livestock. However, the sex-sorting process results in sperm membrane and DNA damage that ultimately decrease fertility. We hypothesized that given the role of protamines in DNA packaging, protamine deficiency could account, at least partially, for the DNA damage observed following sperm sex sorting. To test this, we compared protamine status between unsexed and sexed spermatozoa from two bulls using the fluorochrome chromomycin A3 (CMA3) and flow cytometry. Then, we assessed embryo development following in vitro fertilization (IVF) using the same sperm treatments. Overall, sperm protamination was not different between sexed and unsexed semen. However, one of the two bulls displayed higher rates of protamine deficiency for both unsexed and sexed semen (P < 0.05). Moreover, unsexed semen from this bull yielded lower blastocyst (P < 0.05) and blastocyst hatching rates than unsexed sperm from the other bull. CMA3-positive staining was negatively correlated with cleavage (R2 85.1, P = 0.003) and blastocyst hatching (R2 87.6, P = 0.006) rates in unsexed semen. In conclusion, while the sex-sorting process had no effect on sperm protamine content, we observed a bull effect for sperm protamination, which correlated to embryo development rates following IVF.

Increased quality of in natura and cryopreserved semen of water buffaloes supplemented with saturated and unsaturated fatty acids from the palm oil industry.

Ruminant energy supplementation with vegetable oils or fats has been standing out worldwide and oil palm processing has been receiving growing interest. This study assessed the effect of supplementation with saturated and unsaturated fatty acids from the palm oil industry on the lipid profile of seminal plasma and of the sperm membrane, as well as on the morphological and functional characteristics of raw and cryopreserved buffalo semen. Twelve purebred Murrah bulls (Bubalus bubalis) were assigned to the experimental groups and fed diets for 120 days with no added lipids (CONT, four bulls), or with an extra amount of 3% lipids from crude palm oil (PALM, four bulls), or from palm oil deodorizer distillate (PODD, four bulls). Semen was collected and cryopreserved every 15 days. The lipid composition of membranes and semen quality were determined after collections. Lipid supplementation did not impact feed intake (P>0.05). Diet enrichment with PALM increased the linoleic acid (C18:2,ω6) in seminal plasma. Lipid supplementation did not increase the polyunsaturated fatty acids in the sperm membrane composition, but significantly increased the lignoceric acid (C24:0). Cryopreserved semen of the supplemented bulls presented higher progressive motility (60.2 vs. 67.9 vs. 65.2%; P<0.05) and sperm viability detected by eosin-nigrosin staining (61.1 vs. 69.4 vs. 67.8%; P<0.05). Palm oil reduced major sperm defects in both raw (12.2 vs. 9.3 vs. 13.2%; P<0.0001) and cryopreserved semen (12.4 vs. 9.4 vs. 11.2%; P<0.0001). The lipids added to the diet did not impact the population of spermatozoa with intact plasma and acrosomal membranes (PI-/PSA-), but significantly increased the percentage of spermatozoa with high mitochondrial potential (25.6 vs. 31.5 vs. 32.0%; P=0.008). The results suggest that lipid supplementation based on crude palm oil or palm oil deodorizer distillate can be safely used to feed buffalo bulls and may increase sperm attributes related to male fertility.

In vitro embryo production in buffaloes: from the laboratory to the farm.

Transvaginal follicular aspiration technique together with in vitro embryo production are the biotechnological alternatives currently available to support genetic improvement breeding programs in buffalo species. However, aspects related to animal management, lack of knowledge of the metabolic needs and biochemical peculiarities of gametes and embryos, as well as the reproductive physiology characteristics have hampered progress in the results. Despite the low availability of good quality oocytes collected after OPU in donors as a physiological characteristic of buffalo species, high rates of oocyte maturation, modest embryo cleavage, blastocyst production and pregnancy rates after transvaginal embryo transfer in recipients could be obtained in buffalo in vitro embryo production programs. The results of implementing an in vitro embryo production program in buffaloes in the northern region of Pará state, Brazil, and results published by other groups demonstrate the feasibility of implementing this biotechnology in the routine of breeding programs. Nevertheless, in order to achieve better and consistent results, it is necessary to deepen the knowledge on the peculiarities of reproductive biology in this specie. Selection of donor animals based on ovarian size and ovarian follicular reserve and on the rate of blastocyst production is presented as an effective alternative to increase the efficiency of the in vitro embryo production technique applied to the buffalo species.

Contributions of RNA-seq to improve in vitro embryo production (IVP).

In Vitro Embryo Production (IVP) is widely used to improve the reproductive efficiency of livestock animals, however increasing the embryo development rates and pregnancy outcomes is still a challenge for some species. Thus, the lack of biological knowledge hinders developing specie-specific IVP protocols. Therefore, the contributions of RNA-seq to generate relevant biological knowledge and improve the efficiency of IVP in livestock animals are reviewed herein.

Stem cells from adipose tissue improve the time of wound healing in rats

ABSTRACT PURPOSE: To evaluate the Adipose Stem Cells (ACS) therapy efficacy on the time and quality of wound healing process in rats. METHODS: Nine male Wistar rats were randomly distributed into three groups I) 7 days of healing; II) 14 days of healing; III) 21 days of healing. Four incisions were made on the dorsal surface of each rat and then treated with intralesional ACS, meloxicam, and no treatment and ACS+meloxicam. Macroscopic evaluation was measured by percentage of healing and histopathological by hematoxylin-eosin was performed. RESULTS: All groups have the wound reduced during the three weeks (p<0.001) and after 14 days of healing had greater reduction than others. Wounds treated with ASC had accelerated healing in relation to no treatment and only meloxicam (p<0.001), excepting the ASC+Meloxicam that was similar (p=0.13). There was no difference in histopathological analysis between lesions. CONCLUSION: Adipose stem cell have benefits in reducing time of healing of experimental model of wound in rats, observed 7 days of after application.

Stem cells from adipose tissue improve the time of wound healing in rats.

PURPOSE:: To evaluate the Adipose Stem Cells (ACS) therapy efficacy on the time and quality of wound healing process in rats. METHODS:: Nine male Wistar rats were randomly distributed into three groups I) 7 days of healing; II) 14 days of healing; III) 21 days of healing. Four incisions were made on the dorsal surface of each rat and then treated with intralesional ACS, meloxicam, and no treatment and ACS+meloxicam. Macroscopic evaluation was measured by percentage of healing and histopathological by hematoxylin-eosin was performed. RESULTS:: All groups have the wound reduced during the three weeks (p<0.001) and after 14 days of healing had greater reduction than others. Wounds treated with ASC had accelerated healing in relation to no treatment and only meloxicam (p<0.001), excepting the ASC+Meloxicam that was similar (p=0.13). There was no difference in histopathological analysis between lesions. CONCLUSION:: Adipose stem cell have benefits in reducing time of healing of experimental model of wound in rats, observed 7 days of after application.

Effect of cortisol on bovine oocyte maturation and embryo development in vitro.

Glucocorticoids (GCs) are important mediators of key cellular events. Herein, we investigated the effect of adding cortisol to the IVM medium on the acquisition of developmental competency in bovine oocytes. Cortisol (0.01, 0.1, or 1 µg/mL) had no effect on cleavage rates or cell numbers of resulting blastocysts; however, supplementation with 0.1 µg/mL during IVM increased blastocyst rates of in vitro-fertilized bovine oocytes as compared to untreated controls (41 ± 10% vs. 21 ± 1.2%, P < 0.05, respectively). This concentration was chosen to assess changes in the relative expression of potential GC target genes. Oocytes matured in the presence of cortisol and their corresponding cumulus cells did not show changes in expression for genes analyzed as compared to untreated controls. Notably, blastocysts from oocytes matured in cortisol-supplemented medium expressed higher relative levels of glucose transporter 1 (GLUT1), fatty acid synthase (FASN), and heat shock protein 70 (HSP70). This study supports a role for cortisol in the acquisition of bovine oocyte competence. This is evidenced by increased blastocyst development rates and presumably related to elevated embryonic transcripts with roles in glucose and lipid metabolism, as well as the cellular response to stress.

Environmental quality of an estuary in Amazon delta using immunohistochemical and morphological analyses of gill as biomarkers / Qualidade ambiental de um estuário no delta amazônico utilizando análises imunohistoquímicas e morfológicas de brânquias como biomarcadores

In this study, histological, ultrastructural, and immunohistochemical observations of the gill tissue of two fish species (Plagioscion squamosissimus and Lithodoras dorsalis) were used as biomarkers to assess the ecological conditions of an estuary in the Amazon delta, by relating the health status of the fishes to environmental quality. The gill tissue was analyzed both qualitatively and semi-quantitatively. Marked differences in the type and severity of lesions were verified between species and among study sites. A variety of severe damages were confirmed histologically in specimens from the two impacted areas (B and C), including aneurysms, epithelial lifting, and intense hyperplasia. Electron microscopy and immunohistochemical analyses also revealed injuries in the gill tissue of specimens from these two areas. While, on area A only few specimens showed mild changes in gill tissues and rare marking of apoptosis. These results confirm the urgent need for the more effective control of the discharge of pollutants into the estuary as well as more effective urban planning, given the close proximity of a major industrial complex to residential areas.

Neste estudo foram utilizados biomarcadores histológicos, ultraestruturais e imunohistoquímicos em tecidos brânquiais de duas espécies de peixes, Plagioscion squamosissimus e Lithodoras dorsalis, para avaliar a situação ecológica em um estuário amazônico relacionando a saúde do peixe com a qualidade ambiental. O tecido branquial foi analisado qualitativamente e semiquantitativamente. Foram verificadas diferenças entre as espécies selecionadas e as áreas de estudo, no que se refere ao tipo e severidade das lesões no tecido branquial. Foi detectada uma variedade de alterações histopatológicas severas somente nos peixes capturados nas áreas B e C, tais como: Aneurisma, elevação epitelial e intensa hiperplasia. As análises de microscopia eletrônica e de imunohistoquímica confirmaram danos mais prejudiciais nas brânquias apenas nestas áreas de estudo. Enquanto que na área A poucos peixes apresentaram alterações leves nos tecidos e pouca marcação de apoptose. Estes resultados confirmam a necessidade de um efetivo controle dos dejetos de poluentes assim como um planejamento urbano eficaz nesta região, uma vez que esta área possui um complexo industrial importante próximo à área urbana.

Supplementation of bovine embryo culture medium with L-arginine improves embryo quality via nitric oxide production.

Nitric oxide (NO) is a cell-signaling molecule that regulates a variety of molecular pathways. We investigated the role of NO during preimplantation embryonic development by blocking its production with an inhibitor or supplementing in vitro bovine embryo cultures with its natural precursor, L-arginine, over different periods. Endpoints evaluated included blastocyst rates, development kinetics, and embryo quality. Supplementation with the NO synthase inhibitor N-Nitro-L-arginine-methyl ester (L-NAME) from Days 1 to 8 of culture decreased blastocyst (P < 0.05) and hatching (P < 0.05) rates. When added from Days 1 to 8, 50 mM L-arginine decreased blastocyst rates (P < 0.001); in contrast, when added from Days 5 to 8, 1 mM L-arginine improved embryo hatching rates (P < 0.05) and quality (P < 0.05) as well as increased POU5F1 gene expression (P < 0.05) as compared to the untreated control. Moreover, NO levels in the medium during this culture period positively correlated with the increased embryo hatching rates and quality (P < 0.05). These data suggest exerts its positive effects during the transition from morula to blastocyst stage, and that supplementing the embryo culture medium with L-arginine favors preimplantation development of bovine embryos.

Influence of L-arginine during bovine in vitro fertilization.

The objective of this work was to evaluate the effect of using L-arginine during in vitro fertilization (IVF) on in vitro embryonic development using Bos taurus and Bos indicus semen. Effect of different concentrations (0, 1, 10 and 50 mM) of L-arginine, added to the IVF medium, was evaluated on the fertilization rate at 18 h post-fertilization (hpf), NO3(-)/NO2(-) production during IVF by the Griess colorimetric method (30 hpf), cleavage and blastocyst rates (on Day 2 and Day 7 of culture, respectively) and total blastocyst cell number (Day 7 of culture). The results reveal that the addition of 50 mM L-arginine to IVF medium, with either Bos taurus or Bos indicus spermatozoa, decreased the cleavage rate and blastocyst rate compared to the control group. Other concentrations did not affect embryo production. However, 1 mM L-arginine with Bos indicus semen increased the proportion of hatched blastocysts. These results indicate that high L-arginine concentrations may exhibit toxic effects on bovine gametes during in vitro fertilization.

Expression of PLIN2 and PLIN3 during oocyte maturation and early embryo development in cattle.

In vitro-produced embryos store high lipid content in cytoplasmic lipid droplets (LD), and reduction or removal of LD has been demonstrated to improve freeze-thaw viability. The Perilipin Adipophilin Tail-interacting Protein of 47 kD (PAT) family of proteins is involved in the formation and regulation of LD in many cell types, but their presence has not been addressed either in cattle oocytes or preimplantation embryos. Therefore, this study aimed to detect the expression of PAT family transcripts (Perilipin-2 [PLIN2] and Perilipin-3 [PLIN3]) in immature and in vitro-matured (IVM) oocytes, and in in vitro-produced embryos at the stages of two to four cells, eight to 16 cells, morulae (MO), and blastocyst (BL). The expression of PLIN3 was downregulated in response to IVM, and PLIN2 was comparatively more expressed than PLIN3 in IVM oocytes (P < 0.001). During the early stages of embryo development, PLIN2 expression reached its peak at the MO stage (P < 0.001) and decreased again at the BL stage. In contrast, PLIN3 was expressed in low levels during the earliest stages of development, slightly upregulated at the MO stage (P < 0.05), and greatly increased its expression at the BL stage (15-fold; P < 0.001). PLIN3 was comparatively more expressed than PLIN2 during embryo culture in most stages analyzed (P < 0.05), except in eight- to 16-cell embryos. These results indicate that PLIN2 might be involved in the maintenance of lipid stocks necessary to support embryo development after fertilization of IVM oocytes. Also, we hypothesize that PLIN3 is the main PAT protein responsible for stabilization of LD formed in consequence of the acute lipid load seen during embryo development. We confirmed the presence of both PLIN2 and PLIN3 proteins in BL at Day 7 using immunocytochemistry: these PAT proteins colocalized with LD stained with BODIPY. PLIN3 seemed to be more ubiquitously spread out in the cytoplasm than PLIN2, consistent with the pattern seen in adipocytes. These findings suggest that both elderly (bigger) and newly formed (smaller) LD, positive for PLIN2 and PLIN3 respectively, coexist in blastocysts. To our knowledge this is the first report showing that transcripts of the PAT family are present in cattle oocytes and embryos.

Branchial histopathological study of Brachyplatystoma rousseauxii ( Castelnau, 1855 ) in the Guajará bay, Belém, Pará State, Brazil / Estudo histopatológico da brânquia de Brachyplatystoma rousseauxii na baía do Guajará, Belém, Estado do Pará, Brasil

This work analyzes the morphological alterations in Brachyplatystoma rousseauxii gills and evaluates whether this species can be considered an environmental monitoring biomarker of Guajará Bay. Sampling was carried out in four areas around Belém, Brazil, in four annual periods: dry/wet season, wet season, wet/dry season and dry season. Water pH, temperature and suspended material were evaluated. A total of 36 specimens were collected. The second right gill arch of each animal was removed and immediately fixed and processed for histopathology analysis with light microscopy. The physicochemical analysis of the water during the study period showed slight acidity, temperature and material in suspension within normal levels. Histopathological analysis of the gills from 14 individuals from area I presented no alterations, and only 2 individuals from this area presented some significant type of alteration. In contrast, all individuals captured in areas II, III, and IV presented at least one of the following alterations: aneurism-like alterations, epithelial elevation, infiltration, cell proliferation and cell hypertrophy. Based on the gill histopathological analysis, this organ is considered a good biomarker and the native species B. rousseauxii could be used as a bioindicator for environmental monitoring.

O objetivo deste trabalho foi analisar as alterações morfológicas branquiais em Brachyplatystoma rousseauxii e verificar se esta espécie pode ser considerada como indicador biológico para o monitoramento ambiental da baía do Guajará. As coletas aconteceram em quatro áreas ao redor da cidade de Belém: I (controle), II, III e IV (forte influência antrópica), nos quatro períodos anuais: estação seco/chuvoso, estação chuvoso, estação chuvoso/seco e estação seco. Nesses períodos foram medidos pH, temperatura e material em suspensão. Foram capturados 36 exemplares. O segundo arco branquial direito de cada animal foi retirado, imediatamente fixado e processado para análise histopatológica. A análise físico-química da água revelou pH levemente ácido, a temperatura e o material em suspensão se encontravam nos parâmetros de normalidade. As análises histopatológicas das brânquias de 14 animais da área I não apresentaram alterações branquiais e somente dois revelaram algum tipo de alteração. Todos os indivíduos capturados nas áreas II, III e IV apresentaram pelo menos uma das seguintes alterações: aneurisma, elevação epitelial, infiltração, proliferação celular, fusão lamelar e hipertrofia celular. De acordo com as análises histopatológicas branquiais, este órgão é considerado como um bom biomarcador e a espécie nativa B. rousseauxii pode ser utilizada como bioindicador no monitoramento ambiental.

Cinética da maturaçäo nuclear in vitro de oócitos bubalinos / Nulear maturation kinetcs in vitro of buffalo oocytes

Com o objetivo de avaliar a cinética da maturaçäo in vitro de oócitos bubalinos, foram cultivados 1.619 oócitos com cumulus oophorus compacto em quatro diferentes tratamentos: T1 - TCM 199 com 10 por cento de Soro Fetal Bovino (SFB) (meio base); T2 - Meio base e células da granulosa (CG); T3 - Meio base, CG, 10 UI de gonadotrofina coriônica equina (eCG) e 10 UI de gonadotrofina coriônica humana (hCG); T4 - Meio base, 10 UI de eCG, 10UI de hCG e 1 µg de 17b-estradiol / ml, em tempos de 14 a 17h, 20 a 22h, 23 a 25h, 26 a 28h, 29h e 32h de cultivo em estufa de CO2 a 5 por cento e temperatura de 38,5ºC. No tratamento 1, observou-se que 81,83 por cento, 69,61 por cento, 65,88 por cento, 60,28 por cento, 64,98 por cento e 67,67 por cento dos ovócitos reiniciaram a meiose. No tratamento 2, as taxas de reinicio da meiose foram de 76,06 por cento, 72,06 por cento, 69,78 por cento, 68,12 por cento,74,97 por cento e 89,96 por cento, no tratamento 3 estes percentuais foram de 83,59 por cento, 78,86 por cento, 77,49 por cento, 82,4 por cento, 72,92 por cento e 81,06 por cento e com o tratamento 4 os índices foram de 95,0 por cento, 91,82 por cento, 93, 17 por cento, 91,65 por cento, 92,09 por cento e 74,99 por cento nos tempos de 14 a 17h, 20 a 22h, 23 a 25h, 26 a 28h, 29h e 32h de cultivo, respectivamente, podendo-se concluir que o meio com maior suplementaçäo proporciona maior taxa de reinício da meiose em menor tempo, assim como o aumento no tempo de cultivo pode levar a maiores índices de degeneraçäo