Differential expression of insulin-like growth factor family members in immature cumulus-oocyte complexes from dairy cows with different genotypes.

It has been evident the improvement of in vitro embryo production (IVEP) in dairy cows. Nevertheless, it is known that differences in the number and quality of oocytes between taurine and zebu females impact the efficiency and economic viability of IVEP. As the insulin-like growth factor (IGF) system is related to follicular and oocyte development, we aimed to quantify mRNA abundance of IGF system members and pregnancy-associated plasma protein-A (PAPPA) in the cumulus-oocyte complexes (COCs) of Gir, 1/2 Holstein × 1/2 Gir and Holstein cows. Four pools of 30 immature COCs from Gir, 1/2 Holstein × 1/2 Gir and Holstein cows were obtained by ovum pickup (OPU), and the oocytes and cumulus cells (CC) were mechanically separated and stored at -80°C. Total RNA was extracted from pools of 30 oocytes and their respective CC. Expression of target genes was assessed by real-time RT-PCR. In oocytes, the abundance of IGFR1 mRNA was higher (p < .05) in Gir cows compared with the other breeds. In contrast, in CC, mRNA encoding IGF2 (p < .05), IGFR2 (p < .05) and IGFBP4 (p < .01) was higher in Holstein donors compared with Gir and 1/2 Holstein × 1/2 Gir cows. Additionally, the abundance of PAPPA mRNA was higher in oocytes (p < .001) and CC (p < .01) in Gir and 1/2 Holstein × 1/2 Gir cows compared with the Holstein donors. In conclusion, the higher abundance of PAPPA mRNA in the oocytes and CC from Gir and cross-breed donors combined with the low expression of IGFBP4 in the CC suggests an enhancement of the bioavailability of IGF-free when compared with Holstein COCs.

Lipid profiles of follicular fluid from cows submitted to ovarian superstimulation.

Ovarian superstimulation with exogenous gonadotropins has been extensively used to produce in vivo-derived embryos for embryo transfer in cattle. This process modifies the antral follicle microenvironment and affects oocyte and embryo quality as well the differentiation of granulosa cells. Lipids play significant roles in the cell, such as energy storage, cell structure, and fine-tuning of the physical properties and functions of biological membranes. The phospholipid (PL) contents as well as the effects of superstimulatory treatments on the PL profile of follicular fluid from cows, however, remain unknown. Therefore, to gain insight into the effects of superstimulation with follicle-stimulating hormone (FSH; P-36 protocol) or FSH combined with equine chorionic gonadotropin (eCG; P-36/eCG protocol) on the profile and abundance of PL from cows submitted or not submitted to superstimulatory protocols, were treated with these two superstimulatory protocols. As a control, non-superstimulated cows were only submitted to estrous synchronization. The follicular fluid was aspirated, the remaining cells removed and the follicular fluid stored at -80 °C until extraction. The lipid screening was performed by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) and this technique allowed the identification of sphingomyelins (SM) and phosphatidylcholines (PC) and phosphoethanolamines (PE). The relative abundance of the ions observed in the three experimental groups was analyzed by multivariate and univariate statistical models. The phospholipid SM (16:0) and PC (36:4) and/or PC (34:1) were less (P < 0.05) abundant in the P-36 group compared to the control or P-36/eCG groups. However, the PC (34:2) was more (P < 0.05) abundant in both group of superstimulated cows compared to the control. In summary, ovarian superstimulation seems to modulate the PL content of bovine follicular fluid with a significant increase in PC (34:2), which jointly with others PC and SM, seems to offer a suitable biomarker involved with reproductive processes successful as ovary superstimulation response and embryo development.

Gene expression profile in heat-shocked Holstein and Nelore oocytes and cumulus cells.

The present study determined the transcriptome profile in Nelore and Holstein oocytes subjected to heat shock during IVM and the mRNA abundance of selected candidate genes in Nelore and Holstein heat-shocked oocytes and cumulus cells (CC). Holstein and Nelore cows were subjected to in vivo follicle aspiration. Cumulus-oocyte complexes were assigned to control (38.5°C, 22h) or heat shock (41°C for 12h, followed by 38.5°C for 10h) treatment during IVM. Denuded oocytes were subjected to bovine microarray analysis. Transcriptome analysis demonstrated 127, nine and six genes were differentially expressed between breed, temperature and the breed×temperature interaction respectively. Selected differentially expressed genes were evaluated by real-time polymerase chain reaction in oocytes and respective CC. The molecular motor kinesin family member 3A (KIF3A) was upregulated in Holstein oocytes, whereas the pro-apoptotic gene death-associated protein (DAP) and the membrane trafficking gene DENN/MADD domain containing 3 (DENND3) were downregulated in Holstein oocytes. Nelore CC showed increased transcript abundance for tight junction claudin 11 (CLDN11), whereas Holstein CC showed increased transcript abundance for antioxidant metallothionein 1E (MT1E) . Moreover, heat shock downregulated antioxidant MT1E mRNA expression in CC. In conclusion, oocyte transcriptome analysis indicated a strong difference between breeds involving organisation and cell death. In CC, both breed and temperature affected mRNA abundance, involving cellular organisation and oxidative stress.

Análise da variabilidade da frequência cardíaca em cordeiros da raça Bergamácia, do nascimento aos 35 dias de idade / Analysis of heart rate variability in Bergamasca newborn lambs from birth at 35th days of age

A atividade do sistema nervoso autônomo sobre o coração pode ser verificada pela variabilidade da frequência cardíaca (VFC), método que quantifica e assim infere sobre a modulação autonômica cardíaca, refletindo o funcionamento do sistema nervoso autônomo. Durante o período neonatal, diferenças significativas no que se refere à maturação autonômica são descritas em diversas espécies. Embora a espécie ovina venha sendo utilizada como modelo experimental em diversos protocolos em neonatologia e cardiologia em humanos, estudos descritivos sobre a VFC utilizando animais saudáveis e não sedados são escassos na literatura. O objetivo do estudo foi descrever a VFC em cordeiros da raça Bergamácia durante os primeiros 35 dias de idade. Foram realizados exames eletrocardiográficos em 20 cordeiros da raça Bergamácia a partir do nascimento e semanalmente até o 35° dia de idade. A VFC foi analisada pelo intervalo RR normal (batimentos sinusais), pela frequência cardíaca, pelo índice de tônus vasovagal (iTVV), pela raiz quadrada da média do quadrado das diferenças entre intervalos RR normais adjacentes (RMSSD) e pela raiz quadrada da somatória do quadrado das diferenças dos valores individuais em relação ao valor médio, dividido pelo número de iR-R em 90 segundos - VFC global (RMSM). Todos os parâmetros se alteraram ao longo das semanas. A frequência cardíaca média elevou-se entre o nascimento e os primeiros sete dias de idade, com decréscimo nas quatro semanas subsequentes, sendo o menor valor encontrado aos 35 dias de idade (145,63±37,80bpm). Entre 21, 28 e 35 dias de idade, o iTVV elevou-se significativamente, o RMSM a partir do 28º dia, e, aos 35 dias, o RMSSD, reflexo da ativação parassimpática, exibiu diferenças em relação aos momentos subsequentes. O início da predominância parassimpática, refletida nos índices da VFC, particularmente o iTVV, ocorre aos 21 dias de idade. A partir dos 35 dias de idade, os índices RMSM e RMSSD podem ser utilizados como marcadores fidedignos das mudanças nos efeitos simpático e parassimpático sobre o coração de cordeiros.(AU)

Autonomic nervous system activity can be checked by heart rate variability (HRV), method that quantifies and thus infers on cardiac autonomic modulation, reflecting the functioning of the autonomic nervous system. During the neonatal period significant differences as regards the autonomic maturation, are described in various species. Although the sheep come being used as experimental model of several protocols in neonatology and cardiology in humans, descriptive studies about the VFC using healthy animals and not sedated are scarce in the literature. The objective of this study was to describe the HRV in the Bergamasca race lambs during the first 35 days of age. Electrocardiographic examinations were performed in 20 lambs of the Bergamasca race from birth, in the7th, 14th, 21st, 28th, and 35th days of age. The HRV was analyzed by normal RR interval (sinus rate), heart rate, index of vagal tone, and the cardiovascular manifestations (square root of the average of the square of the differences between normal RR intervals adjacent). All parameters have changed over the weeks. The average heart rate increased between the birth and the first seven days of age, with decrease in the subsequent four weeks, being the smallest value found at 35 days of age (145.63±37.80bpm). Between 21, 28 and 35 days of age, the iTVV increased significantly, the RMSM from the 28 day, and 35 days, the cardiovascular manifestations, reflex parasympathetic activation, exhibited differences in relation to subsequent times. The beginning of parasympathetic dominance, reflected in the HRV indices, particularly the iTVV, occurs at 21 days of age. From the 35 days of age, the RMSM indices and RMSSD can be used as reliable markers of change in sympathetic and parasympathetic effect on the heart of lambs.(AU)

Effect of pregnancy and correlation of weight and heart rate with electrocardiographic parameters in the American Miniature Horse / Efeito da prenhez e correlação do peso e frequência cardíaca com os parâmetros eletrocardiográficos de equinos Mini-horse

There are studies on electrocardiogram (ECG) in several breeds of horses, which highlights the growing importance of cardiology in this species. But few authors have addressed the influence of pregnancy on the cardiac physiology of the mare, and specifically, there are no studies on the Mini-horse breed, as well as no correlation of body weight (BW) and heart rate (HR) with electrocardiographic parameters in this breed. The aims of this study were to determine the effects of pregnancy on ECG parameters and to examine the relationships between these variables and body weight (BW) and heart rate (HR). A total of 203 animals were used, including 143 females (66 pregnant) and 60 males. Electrocardiographic examinations were performed by computerized electrocardiogram (TEB), and the parameters were evaluated in six leads in the frontal plane (Lead I, II, III, aVR, aVL and aVF) and base-apex (BA). BW was inversely proportional to HR, which in turn showed an inverse relationship with the duration of the P-wave and the PR and QT intervals. The P-wave amplitude (lead II) was higher in pregnant animals than in non-pregnant animals. The effect of reproductive status should be monitored by ECG throughout the entire gestational period. The 66 pregnant mares in this study were examined during the first third of their pregnancies, thus, few differences were observed between pregnant and non-pregnant animals.(AU)

Existem estudos sobre eletrocardiograma (ECG) em diversas raças de equinos, o que ressalta a crescente relevância da cardiologia nessa espécie. Porém, poucos autores abordaram a influência da gestação na fisiologia cardíaca da égua, e, especificamente, não há trabalhos sobre a raça Mini-horse, assim como não há correlação do peso vivo (PV) e frequência cardíaca (FC) com os parâmetros eletrocardiográficos nessa raça. O objetivo deste estudo foi estudar a influência da prenhez sobre os parâmetros eletrocardiográficos, além de verificar a correlação do PV e FC com essas variáveis. Utilizaram-se 203 animais dessa raça, hígidos, sendo 143 fêmeas (66 prenhes) e 60 machos. Os exames eletrocardiográficos foram realizados por eletrocardiograma computadorizado (TEB), e os parâmetros foram avaliados em seis derivações do plano frontal (I, II, III, aVR, aVL e aVF) e base-ápice. O PV foi inversamente proporcional à FC, que também mostrou relação inversa com a duração da onda P e os intervalos PR e QT. Na derivação II, a amplitude da onda P foi maior nos animais prenhes do que vazios. No entanto, sugere-se que a influência do estado reprodutivo seja estudada de forma progressiva pelo acompanhamento de todo o período gestacional, visto que as 66 éguas estudadas se apresentavam no terço inicial da prenhez, portanto poucas diferenças foram observadas entre animais prenhes e vazios.(AU)

Concentração de lactato sérico em ovelhas e cordeiros mestiços (1/2 Suffolk) nascidos em eutocia / Lactate blood levels in sheep and newborn lambs (1/2 suffolk) in normal delivery

O objetivo do estudo foi avaliar a concentração de lactato sanguíneo venoso em ovelhas e em cordeiros neonatos imediatamente após o parto eutócico. Foram utilizadas 14 ovelhas, sendo nove da raça Suffolk e quatro mestiças e 20 cordeiros neonatos sadios (machos e fêmeas). O efeito do sexo, da raça e do número de cordeiros por parto foi investigado, bem como a possível correlação entre a concentração de lactato materno e neonatal. Não houve diferença entre machos e fêmeas, em relação à raça (Suffolk e mestiços), contudo a concentração de lactato foi mais elevada em cordeiros gêmeos ou trigêmeos quando comparado a cordeiros uníparos. Houve correlação positiva entre a concentração materna e a neonatal, sendo que quanto maior a concentração de lactato materno, maior a concentração no cordeiro imediatamente após o parto eutócico. Os resultados do presente estudo fornecem um subsídio por meio do qual se podem comparar os níveis de lactato sanguíneo em cordeiros em estado crítico.

The aim of this study was to evaluate the venous blood lactate concentration in sheep and newborn lambs immediately after normal delivery. We used 14 ewes, nine and four Suffolk crossbred lambs and 20 healthy newborns (males and females). The effect of gender, race and number of lambs per birth was investigated, as well as the possible correlation between the concentration of maternal and newborn lactate. There was no difference between males and females or in relation to race (and Suffolk crossbred); however the concentration of lactate was higher in twin or triplet lambs when compared to single lambs. There was a positive correlation between the maternal and newborn concentration and the higher the concentration of lactate, the greater the concentration in the lamb immediately after normal delivery. The results of this study provide a base from which to compare the blood lactate levels in lambs in critical condition.

In vitro cultivation of canine multipotent mesenchymal stromal cells on collagen membranes treated with hyaluronic acid for cell therapy and tissue regeneration

Support structures for dermal regeneration are composed of biodegradable and bioresorbable polymers, animal skin or tendons, or are bacteria products. The use of such materials is controversial due to their low efficiency. An important area within tissue engineering is the application of multipotent mesenchymal stromal cells (MSCs) to reparative surgery. The combined use of biodegradable membranes with stem cell therapy may lead to promising results for patients undergoing unsuccessful conventional treatments. Thus, the aim of this study was to test the efficacy of using membranes composed of anionic collagen with or without the addition of hyaluronic acid (HA) as a substrate for adhesion and in vitro differentiation of bone marrow-derived canine MSCs. The benefit of basic fibroblast growth factor (bFGF) on the differentiation of cells in culture was also tested. MSCs were collected from dog bone marrow, isolated and grown on collagen scaffolds with or without HA. Cell viability, proliferation rate, and cellular toxicity were analyzed after 7 days. The cultured cells showed uniform growth and morphological characteristics of undifferentiated MSCs, which demonstrated that MSCs successfully adapted to the culture conditions established by collagen scaffolds with or without HA. This demonstrates that such scaffolds are promising for applications to tissue regeneration. bFGF significantly increased the proliferative rate of MSCs by 63% when compared to groups without the addition of the growth factor. However, the addition of bFGF becomes limiting, since it has an inhibitory effect at high concentrations in culture medium.

Concentração de lactato e glicemia em cadelas e neonatos nascidos de cesariana / Lactato concentration in bitch and canine neonates born through cesarean section

Avaliaram-se a concentração de lactato e a glicemia em sete cadelas em trabalho de parto submetidas à cesariana e em seus respectivos neonatos. O lactato materno foi mensurado utilizando-se medidor portátil e pelo método enzimático colorimétrico. Nos neonatos avaliou-se também o peso ao nascimento. Os valores de lactato materno e neonatal foram estatisticamente diferentes, sendo mais elevado nos neonatos. Ao nascimento, as cadelas e os neonatos encontravam-se normoglicêmicos, e não se observou correlação entre a glicemia de ambos. Os valores de lactato obtidos pelo o analisador portátil (3,5±0,6mmol/L) foram mais altos do que os obtidos pelo método enzimático colorimétrico (1,6±0,6mmol/L). Observou-se correlação significativa entre o peso do neonato e a concentração de lactato, isto é, os filhotes de menor peso apresentaram maior concentração de lactato.

This work evaluated the concentration of lactate and blood glucose in seven bitches in labor subject to caesarean section and in their newborns. The lactate concentration was measured using a portable analyzer and enzymatic colorimetric method. In neonates, the birth weight was evaluated. The maternal and neonatal lactate values were statistically different, being higher in newborns. At birth, the bitches and the newborns had normal glucose status, and no correlation was observed between blood glucose in either. The lactate concentration obtained by the portable analyzer (3.5±0.6mmol/L) was higher than that obtained with the enzymatic colorimetric method (1.6±0.6mmol/L). There was significant correlation between the neonate weight and lactate concentration and in puppies with lighter weight there was a higher concentration of lactate.

Estudo da pressão arterial pelo método indireto oscilométrico (petmap®) em cães domésticos não anestesiados / Study of blood pressure by the indirect oscillometric method (petmap®) in domestic unanesthetized dogs

O estudo das pressões arteriais sistólica, média, diastólica e da frequência cardíaca, pelo método indireto oscilométrico (petmap®), foi realizado em 150 cães atendidos pelo Serviço de Clínica Médica de Pequenos Animais da FMVZ - Unesp/Botucatu. Investigou-se a influência de fatores como presença do proprietário, estado de saúde, diagnóstico de doença renal, raça, idade, sexo, decúbito, contenção, fluidoterapia, condição corpórea, temperamento, atividade física, dieta e atitude associados ou não à elevação da pressão arterial. Dos 150 cães, 34% encontravam-se sob a categoria de risco mínimo para o desenvolvimento de lesões hipertensivas, 14,6% com hipertensão branda, 22,6% com hipertensão moderada e 28,66%, com hipertensão grave. Houve influência, dos fatores analisados, na elevação da pressão arterial de acordo com a categoria de risco.

The study of systolic, mean, diastolic and heart rate by the oscillometric indirect method (PetMapTM) was performed in 150 dogs attended by the Small Animal Service of Medicine of FMVZ - Unesp/Botucatu. The influence of factors such as presence of the owner, health status, diagnosis of kidney disease, race, age, sex, recumbency, containment, fluid, body condition, temperament, physical activity, diet and attitude associated or not with elevated blood pressure was investigated. Of the 150 dogs, 34% were under the category of minimal risk for the development of hypertensive lesions, 14.6% with mild hypertension, 22.6% with moderate hypertension and 28.66% with severe hypertension. The analyzed factors influenced the elevation of blood pressure according to risk category.

In vitro cultivation of canine multipotent mesenchymal stromal cells on collagen membranes treated with hyaluronic acid for cell therapy and tissue regeneration.

Support structures for dermal regeneration are composed of biodegradable and bioresorbable polymers, animal skin or tendons, or are bacteria products. The use of such materials is controversial due to their low efficiency. An important area within tissue engineering is the application of multipotent mesenchymal stromal cells (MSCs) to reparative surgery. The combined use of biodegradable membranes with stem cell therapy may lead to promising results for patients undergoing unsuccessful conventional treatments. Thus, the aim of this study was to test the efficacy of using membranes composed of anionic collagen with or without the addition of hyaluronic acid (HA) as a substrate for adhesion and in vitro differentiation of bone marrow-derived canine MSCs. The benefit of basic fibroblast growth factor (bFGF) on the differentiation of cells in culture was also tested. MSCs were collected from dog bone marrow, isolated and grown on collagen scaffolds with or without HA. Cell viability, proliferation rate, and cellular toxicity were analyzed after 7 days. The cultured cells showed uniform growth and morphological characteristics of undifferentiated MSCs, which demonstrated that MSCs successfully adapted to the culture conditions established by collagen scaffolds with or without HA. This demonstrates that such scaffolds are promising for applications to tissue regeneration. bFGF significantly increased the proliferative rate of MSCs by 63% when compared to groups without the addition of the growth factor. However, the addition of bFGF becomes limiting, since it has an inhibitory effect at high concentrations in culture medium.