Determination of intraocular pressure and Schirmer tear test and the comparison between the applanation tonometer (Tono-Pen AVIA®) and the rebound tonometer (TonoVet Plus®) in mini lionhead rabbits.

OBJECTIVE: To determine intraocular pressure (IOP) and tear production, as well as to compare the IOP obtained with the TonoVet Plus® (rebound) with the Tono-Pen Avia® (applanation) tonometers. ANIMALS: Twenty-five Mini Lionhead rabbits (n = 50 eyes). PROCEDURE: Tear production was measured at 6:00 a.m. and 6:00 p.m. by using the STT. The IOP reading was performed with the rebound tonometer, followed by the applanation tonometer, at 6:00 a.m., 9:00 a.m., 12:00 p.m., 3:00 p.m., and 6:00 p.m. Regression analysis, analysis of variance (anova) and Bland-Altman statistics were used. RESULTS: Daily tear production was 10.25 ± 3.75 mm/min, with no differences among the moments evaluated. Average daily IOP was 17.7 ± 3.08 mmHg with the TonoVet Plus® and 11.5 ± 4.56 mmHg with the Tono-Pen Avia®. IOP values were higher at the beginning and end of the day with both tonometers. CONCLUSION: The IOP values are higher with the TonoVet Plus® tonometer. The reference values of IOP and tear production obtained in this work may support the diagnosis, treatment, and monitoring of ocular disorders in pet Mini Lionhead rabbits.

Methionine sources at different dietary levels alters the growth and expression of genes related to homocysteine remethylation in the jejunum of broilers.

Sulfur amino acids are essential for the proper development of broilers and are required throughout the bird's life to perform important physiological functions. Studies that seek to understand the actions of sulfur amino acids in the body of birds are essential. The present study evaluated the influence of sulfur amino acid supplementation using DL-Methionine (DL-Met) and DL-Methionine hydroxy analogue (DL-HMTBA), on the performance and expression of genes related to methionine metabolism, in the jejunum of broilers. Four hundred and fifty male broilers (Cobb-700 slow feathering) were distributed in a completely randomized design, in a factorial scheme (2x3), with two sources of methionine (DL-Met and DL-HMTBA) and three levels of methionine (deficiency, requirement and excess). The mRNA expression of the MAT1, MTR, BHMT, MTRR, CBG and GSS genes, and performance data such as feed intake, weight gain, and feed conversion were evaluated. DL-HMTBA increased the expression of BHMT (p = 0.0072) and MTRR (p = 0.0003) in the jejunum of the birds. Methionine deficiency increased the expression of BHMT (p = 0.0805) and MTRR (p = 0.0018). Higher expression of GSS was observed in birds that were supplemented with DL-HMTBA (p = 0.0672). Analyzing our results, it is preferable to supplement sulfur amino acids with DL-Met at the requirement level. Birds fed with DL-HMTBA showed worse weight gain (p = 0.0117) and higher feed conversion (p = 0.0170); methionine deficiency resulted in higher feed intake (p = 0.0214), lower weight gain (p<0.0001) and consequently higher feed conversion (p<0.0001). Based on the information found in this work, it is recommended to supplement sulfur amino acids with DL-Met at the level of compliance with the requirement.

Relationship between adiponectin and fertility in the female pig.

Adiponectin isoforms may mediate different aspects of the pleiotropic function of the protein, including the reproductive process. We examined the pattern of circulating adiponectin and adiponectin system expression in fat and ovarian tissues of hyperfertile and subfertile sows. We demonstrated the presence of five different isoforms of adiponectin (90, 158, 180, 250 and >250kDa) in the circulation and identified a subgroup of subfertile females that displayed reduced abundance of all adiponectin isoforms as well as a lack of the 250-kDa adiponectin isoform in both serum and follicular fluid. Subfertility in these animals was associated with fewer large follicles and corpora lutea in the ovaries, as well as lower concentrations of 17ß-oestradiol in the follicular fluid of large follicles. In addition, subfertile females showed higher adiponectin mRNA in fat tissue and altered mRNA and protein expression of adiponectin and its receptors in the ovary. Changes in the abundance and pattern of circulating adiponectin isoforms have been associated with reproductive disorders in animals and humans, including polycystic ovarian syndrome (PCOS). Our findings suggest that the adiponectin system may play an important role in controlling ovarian function and influencing porcine fertility.

Potencial envolvimento da adiponectina e seus receptores na modulação da esteroidogênese em corpo lúteo de cadelas ao longo do diestro / Potential involvement of adinopectin and its receptors in the modulation of steroidogenesis in corpus luteum of bitches during diestrus

No ciclo estral de cadelas a fase luteínica, denominada diestro, compreende um período que varia de 60 a 100 dias em animais não-prenhes, caracterizado pela elevação plasmática de progesterona nos primeiros 20 dias pós ovulação (p.o). A adiponectina é a mais abundante proteína secretada pelo tecido adiposo, porém sua concentração plasmática diminui significativamente em alterações metabólicas como resistência insulínica e Diabetes mellitus tipo2, alterações descritas como relacionadas em algumas cadelas com o período de diestro. O objetivo do estudo foi determinar a expressão e imunolocalização do sistema adiponectina (adiponectina e seus receptores, adipoR1 e adipoR2) no corpo lúteo de cadelas ao longo do diestro, correlacionando-o ao perfil hormonal de 17β-estradiol e progesterona, assim como à expressão de um dos genes alvo do sistema, o PPAR-γ. Para realização do estudo foram coletados corpos lúteos de 28 cadelas durante ovariosalpingohisterectomia de eleição nos dias 10, 20, 30, 40, 50, 60 e 70 pós ovulação (o dia zero da ovulação foi considerado aquele no qual a concentração plasmática de progesterona atingiu 5ng/mL). Os corpos lúteos foram avaliados por imunohistoquímica para adiponectina e seus receptores e a expressão do RNAm do PPAR-γ por PCR em tempo real. A análise estatística da avaliação gênica foi realizada com o teste ANOVA, seguido por comparação múltipla Newman-Keuls. O sinal da adiponectina apresentou-se mais intenso até os primeiros 20 dias p.o, momento de regência da progesterona; houve queda gradativa após este período, coincidindo com a ascensão do 17β-estradiol, cujo pico foi notado próximo do dia 40 p.o. A queda marcante da adiponectina ocorreu após 50 dias p.o. O sinal do adipoR1 mostrou-se bem evidente até os 40 dias p.o e o do adipoR2 até os 50 dias p. o, decaindo posteriormente. Foi observada maior expressão do gene PPAR-γ aos 10, 30 e 70 dias p.o. Estes resultados mostram que a expressão protéica da adiponectina e de seus receptores se altera ao longo do diestro e que estas alterações podem estar relacionados às alterações hormonais e expressão do PPAR- γ, participando do mecanismo fisiológico de desenvolvimento, manutenção, atividade e regressão luteínica em cadelas.

In the estrous cycle of bitches, the luteal phase or diestrus includes a period ranging from 60 to 100 days in non-pregnant animals, characterized by elevated serum progesterone during the first 20 days post-ovulation (p.o). Adiponectin is the most abundant protein secreted by adipose tissue, but plasma concentration decreases significantly in metabolic disorders like insulin resistance and diabetes mellitus type 2, described as related changes in some bitches in diestrus. The aim of this study was to determine the expression and immunolocalization of the adiponectin system (adiponectin, and adipoR1 adipoR2) in the corpus luteum during diestrus, and correlate it to hormonal profile of 17β-estradiol and progesterone, as well as the expression of a gene target of the system, the PPAR-γ. For the study, corpora lutea were collected from 28 dogs during ovariosalpingohysterectomy on days 10, 20, 30, 40, 50, 60 and 70 post ovulation (day zero of ovulation was considered the day when the plasma progesterone concentration reached 5ng/mL). The corpora lutea were evaluated by immunohistochemistry for adiponectin, adipoR1 and adipoR2 and mRNA expression of PPAR-γ by real-time PCR. Statistical analysis of gene expression was performed with ANOVA followed by Newman-Keuls multiple comparisons. Adiponectin positive signal was stronger during the first 20 days p.o, time of the regency of progesterone; there was a gradual adiponectin and progesterone decline after this period, coinciding with the rise of 17β-estradiol, whose peak was near the 40 days p.o. The markedly adiponectin decrease occurred after 50 days p.o. The signal of adipoR1 was markedly evident at 40 days p.o and that of adipoR2 up to 50 days p.o, declining afterwards. We observed higher expression of PPAR-γ gene at 10, 30 and 70 days p.o. These results show that adiponectin and its receptors protein expression is altered during the diestrus and that these changes may be related to hormonal changes and expression of PPAR-γ, participating in the physiological mechanism of development, maintenance, activity and luteal regression in bitches.

Avaliação morfológica das membranas fetais e da placenta de Mazama gouazoubira (veado-catingueiro) de vida livre no terço inicial da gestação / Morphological evaluation of fetal membranes and placenta of wild life Mazama gouazoubira deer in the first third of pregnancy

Mazama gouazoubira, o veado-catingueiro, é uma espécie de cervídeo de porte pequeno, que pode ser encontrado na América do Sul, desde o sul do Uruguai até o norte de Mato Grosso, no Brasil. Este estudo teve como objetivo descrever as membranas fetais e a placenta de M. gouazoubira no terço inicial de gestação. As amostras coletadas foram analisadas macroscopicamente e microscopicamente. O exame do útero demonstrou uma gestação univitelina e um embrião com crown-rump de 13mm. Na análise do embrião pode ser observado o olho pigmentado, as saliências auriculares, o mesonefro e e metanefro em desenvolvimento, o fígado e sua proeminência externa, o estômago, os membros torácicos e os brotos dos membros pélvicos. A placenta apresentou-se oligocotiledonária e no útero puderam ser observadas nove carúnculas. O saco gestacional mediu 15cm de comprimento e, como observado no início da gestação dos ruminantes domésticos, os cotilédones não puderam ser identificados macroscopicamente. Uma fraca adesão foi observada entre as carúnculas e a membrana corioalantóica (cotilédones) que formavam os placentônios. A membrana corioalantóica demonstrou um alantóide bem vascularizado composto por uma fina camada de células de núcleos e citoplasmas alongados. Na outra face da membrana, o cório foi composto por células cúbicas, de citoplasmas escassos e núcleos grandes e arredondados, características de células trofoblásticas. Envolvendo o embrião visualizou-se o saco amniótico constituído de duas camadas passíveis de separação mecânica, que apresentaram morfologia similar, sendo formadas por um epitélio pavimentoso avascular. Não foi observado saco vitelino no estágio gestacional do espécime estudado. Conclui-se que a placenta de M. gouazoubira é oligocotiledonária, como observado em outros cervídeos, e que as membranas fetais apresentam semelhanças com a de outros ruminantes, incluindo as características citológicas. Estudos adicionais são necessários para determinar a presença do saco vitelino e quando ocorre sua regressão.

Mazama gouazoubira is a small-sized deer, which can be found in South America, from southern Uruguay to the north of Mato Grosso, Brazil. This study aimed to describe the fetal membranes and placenta of M. gouazoubira in the first third of pregnancy. Samples collected were macroscopically and microscopically analyzed. Uterus examination demonstrated univitelinic gestation with an embryo measuring a crown-rump length of 13mm. In the embryo analysis we could identify the pigmented eye, auricular hillocks, mesonephron and early metanephron, liver and its external prominence, stomach, thoracic members and buds of pelvic members. Placenta was oligocotyledonary and nine caruncles were found in the uterus. Gestational sac measured 15cm in length, and, as observed in the early gestation of domestic ruminants, cotyledons could not be identified macroscopically. A weak adhesion was found between caruncles and chorioallantoic membrane (cotyledons) forming the placentomes. The chorioallantoic membrane showed a well-vascularized allantoid composed by a thin layer of cells with stretched nucleus and cytoplasm. On the other side of the membrane, the chorion was composed by cubic cells with scant cytoplasm and large-rounded nuclei, features of trophoblast cells. Involving the embryo we identified a two-layered amniotic sac. Layers could be mechanically separated and had similar morphology, showing a non-vascularized squamous epithelium. There was no yolk sac in the gestational stage of this specimen. We concluded that the placenta of M. gouazoubira is an oligocotyledonary placenta, as observed in others cervids, and that the fetal membranes showed similarities with those from other domestic ruminants, including cytological characteristics. Further studies are required in order to determine the presence of the yolk sac and its regression time.

Produção de progesterona in vitro pelas células do corpo lúteo bovino ao longo da gestação / In vitro progesterone production from bovine corpus luteum throughout gestation

O presente trabalho foi desenvolvido para testar a hipótese de que células luteínicas bovinas em cultivo, provenientes dos três terços de gestação, comportam-se da mesma maneira que células in vivo em relação à produção de P4. Foram coletadas amostras de corpos lúteos (CL) de 90 (n=3), 150 (n=3) e 210 (n=3) dias de gestação obtidos em abatedouro. Sob condições assépticas, as células foram mecanicamente dispersas e cultivadas em placas de 96 poços. Após 24 horas de cultivo foram feitas a lavagem dos poços e a adição do precursor pregnenolona. Os tratamentos foram realizados em octuplicata para cada tempo de tratamento (24, 48 e 96 horas) com três repetições de cada período gestacional. As amostras de meio de cultura e as células foram coletadas 24, 48 e 96 horas após adição do precursor e acondicionadas em freezer a -20ºC até o processamento. A progesterona foi dosada através de radioimunoensaio e o conteúdo protéico pelo método de Lowry. Os resultados foram analisados estatisticamente e considerados diferentes quando p<0.05. Foi observada maior produção de P4 aos 90 dias de gestação (35,277±0,075), posterior decréscimo aos 150 dias (28,820±0,231) e novo aumento aos 210 dias (32,777±0,099). A produção de P4 em células cultivadas por 24 horas foi maior (p<0,05) em células oriundas do grupo de 90 dias (2,912±0,047) quando comparado a 150 (2,669±0,137) e 210 dias (2,741±0,088). As 48 e 96 horas de cultivo, células luteínicas bovinas de 90 dias produziram mais P4 que células de 210 dias (2,934±0,029 e 2,976±0,121 respectivamente x 2,760±0,059 e 2,695±0,149, respectivamente; p<0,05), que por sua vez produziram mais do que células de 150 dias (2,334±0,084 para 48 horas e 2,205±0,136 para 96 horas). Aos 150 dias de gestação a produção de progesterona apresentou diminuição gradativa ao longo das 96 horas de cultivo. Essas diferenças podem ser explicadas pela expressão gênica diferencial de enzimas ou também de fatores presentes na cascata esteroidogênica...

The aim was to test the hypothesis that cultivated bovine luteal cells from three different thirds of pregnancy behave the same way as in vivo luteal cells relative to P4 production. Corpus luteum samples from days 90 (n=3), 150 (n=3) and 210 (n=3) of pregnancy were obtained at a local slaughterhouse. Under aseptic conditions cells were mechanically dispersed and cultivated in a 96 wells-plate. After 24 hours of culture, cells were washed and the precursor pregnenolone was added. Experiments were conducted eight times for each studied time period (24, 48 and 96 h) and three times for each gestational age. Culture medium and cells were collected after 24, 48 and 96 hours of precursor addition and kept frozen at -20ºC until processing. Progesterone was measured by RIA and protein content by Lowry's method. Results were statistically analyzed and considered different when p <0.05. A higher P4 production was observed on day 90 of gestation (35.277±0.075), then this production was decreased at day 150 (28.820±0.231) and increased again at day 210 (32.777±0.099). After 24 hours of culture, luteal cells P4 production reached maximum values in the group of 90 days (2.912±0.047) when compared to 150 (2.669±0.137) and 210 days (2.741±0.088). At 48 and 96 hours of culture, bovine luteal cells from day 90 of gestation produced more P4 than cells from day 210 (2.934±0.029 and 2.976±0.121 respectively x 2.760±0.059 and 2.695±0.149, respectively; p<0.05), which in turn, produced more P4 than cells from day 150 (2.334±0.084 for 48 h and 2.205±0.136 for 96 h). Luteal cells from day 150 of gestation presented a decreasing P4 production throughout the 96 hours of culture. These differences could be explained by differential gene expression of enzymes and/or factors belonging to the esteroidogenic cascade in accordance to the gestational period. The established luteal cell culture model could be used for further functional studies once P4 secretion pattern...

Adiponectin induces periovulatory changes in ovarian follicular cells.

Adiponectin, the most abundantly synthesized protein in adipose tissue, has plieotropic effects on liver, muscle, endothelium, placenta, and other tissues. We examined direct effects of recombinant porcine adiponectin on porcine ovarian granulosa cells in vitro. We demonstrate that adiponectin, at physiologically relevant levels (10-25 microg/ml), provokes expression of genes associated with periovulatory remodeling of the ovarian follicle over a time frame of 6-24 h. These include cyclooxygenase-2, prostaglandin E synthase, and vascular endothelial growth factor. Adiponectin modulates steroid synthetic protein gene expression, increasing steroidogenic acute regulatory protein transcript abundance and reducing cytochrome P450aromatase. Adiponectin has antidiabetic properties and sensitizes tissues to insulin. We show that it interacts with both LH and insulin in inducing expression of cyclooxygenase-2 transcripts in granulosa cells. We determined that the MAPK pathway, via phosphorylation of ERK1/2, is involved in mediation of the adiponectin signal in ovarian granulosa cells, rather than protein kinase A or the classic adiponectin transducer, AMP-activated protein kinase. Adiponectin synthesis is reduced in obesity, and our findings suggest that this reduction plays a role in obesity-related ovarian dysfunction.

Immunohistochemical localization of VEGF and its receptors in the corpus luteum of the bitch during diestrus and anestrus.

The corpus luteum (CL) is a temporary endocrine gland, whose life span depends on the interaction of luteotrophic and luteolytic factors. Since development and maintenance of CL is based on angiogenesis, angiogenic growth factors may play a role in CL-function of the bitch, as described for other species. The aim of this study was to detect the presence of the vascular endothelial growth factor (VEGF) system in the bitch CL throughout diestrus and early anestrus. For that purpose, blood samples from 24 bitches were collected and analyzed for progesterone to determine ovulation time and the animals were subjected to ovariosalpingohysterectomy 10, 20, 30, 40, 50, 60 or 70 days after ovulation. The corpora lutea were fixed in formalin and embedded in Paraplast resin. Five micrometers sections were submitted to standard immunohistochemistry protocol using three primary antibodies (SC-315, SC-316 and VG76e) for detection of kinase domain region (KDR), fms-like tyrosine kinase 1 (Flt-1) and VEGF, respectively. The VEGF system expression could be detected in all diestrus stages in endothelial as well as luteal cells (responsible for blood vessel formation and progesterone production, respectively), indicating time dependent changes: immunostaining tended to increase from Day 10 to 50 and to decrease until Day 70 post-ovulation. In the CL of the bitch, structure related cells, like pericytes and stroma cells, expressed it in determined time points of diestrus with little intensity variation. We concluded that VEGF might have a modulatory effect in the CL of the dog acting as paracrine and autocrine factor through its receptors, Flt-1 and KDR.