In vitro culture of red-rumped agouti preantral follicles enclosed in fresh and vitrified ovarian tissues using TCM199 plus different pFSH concentrations

Considering the relevance of establishing biodiversity conservation tools, the study aimed to investigate the TCM199 supplemented with different follicle-stimulating hormone (FSH) concentrations on survival and development of fresh and vitrified preantral follicles enclosed in red-rumped agouti ovarian tissues cultured in vitro. In the first experiment, six pairs of ovaries were fragmented and cultured for 6 days according to groups: 10 ng/mL pFSH (FSH10 group) and 50 ng/mL (FSH50 group). Non-cultured tissues were considered as a control. In the second experiment, vitrified/warmed fragments of four pairs of ovaries were cultured with the best concentration of FSH established (cryopreserved and cultured group). Non-cryopreserved (fresh control group) and cryopreserved but non-cultured (non-cultured group) tissues were used as controls. For both experiments, preantral follicles were evaluated for survival and development using morphological and viability analysis by trypan blue staining. After culturing fresh samples, FSH50 showed a higher percentage of morphologically normal follicles when compared to FSH10 (P < 0.05). This same response was observed for primordial follicles. Regardless of the concentrations of FSH used during in vitro culture, no difference was observed regarding the percentage of viable follicles and diameters (P > 0.05). Thus, the FSH50 group was used for second experiment, in which 76.2 ± 7.2% normal preantral follicles previously vitrified was found after 6-day culture, also presenting the highest values (P < 0.05) for morphology of primordial follicles (95.2 ± 4.7%). Nevertheless, in vitro culture did not affect the viability and diameter of preantral follicles of cryopreserved tissues (P > 0.05). In conclusion, TCM199 supplemented with 50 ng/mL FSH was efficient in maintaining the in vitro survival of fresh and vitrified red-rumped agouti preantral follicles. This was the first study related to the in vitro culture of ovarian preantral follicles in this species, aiming to contribute to its conservation.(AU)

The ovarian and uterine responses of Baixadeiro mares to prostaglandin synchronization during the dry and rainy seasons

This study aimed to evaluate the effect of synchronization with prostaglandin F2α in Baixadeiro mares during the rainy and dry seasons. Fourteen mares were synchronized by administering two doses of 1 mL prostaglandin PGF 2α and monitored by rectal palpation and ultrasound for the assessment of follicular development and uterine echotexture. Of this total, nine mares allowed the collection of blood, in which the blood was collected by venipuncture of the jugular vein to determine progesterone (P4) by ELISA. Mares showed no differences (P > 0.05) in weight, body score condition (BSC), tone, uterine edema, frequency of ovulation, synchronization interval, estrus, and the total number of follicles between periods. However, there was a difference in large increased follicle diameter (P < 0.05) during the dry season. The average concentrations of P4 in mares differed (P < 0.05) between the pre- and post-ovulatory phases for both seasons and after ovulation, with higher concentrations in the rainy season. Furthermore, statistical differences in daily light (P < 0.05) were observed between the dry and rainy periods. Thus, we conclude that mares from the genetic grouping Baixadeiro showed no reproductive seasonality, though there was a difference in luminosity between the rainy and dry seasons. The treatment with two doses of PGF 2α was effective in synchronizing the mares, promoting the return of estrus in the dry and rainy periods. The mares remaining cyclically active throughout the year provided there were appropriate forage availability and quality levels to allow for normal values of body weight and condition.

The ovarian and uterine responses of Baixadeiro mares to prostaglandin synchronization during the dry and rainy seasons

Abstract This study aimed to evaluate the effect of synchronization with prostaglandin F2α in Baixadeiro mares during the rainy and dry seasons. Fourteen mares were synchronized by administering two doses of 1 mL prostaglandin PGF 2α and monitored by rectal palpation and ultrasound for the assessment of follicular development and uterine echotexture. Of this total, nine mares allowed the collection of blood, in which the blood was collected by venipuncture of the jugular vein to determine progesterone (P4) by ELISA. Mares showed no differences (P > 0.05) in weight, body score condition (BSC), tone, uterine edema, frequency of ovulation, synchronization interval, estrus, and the total number of follicles between periods. However, there was a difference in large increased follicle diameter (P < 0.05) during the dry season. The average concentrations of P4 in mares differed (P < 0.05) between the pre- and post-ovulatory phases for both seasons and after ovulation, with higher concentrations in the rainy season. Furthermore, statistical differences in daily light (P < 0.05) were observed between the dry and rainy periods. Thus, we conclude that mares from the genetic grouping Baixadeiro showed no reproductive seasonality, though there was a difference in luminosity between the rainy and dry seasons. The treatment with two doses of PGF 2α was effective in synchronizing the mares, promoting the return of estrus in the dry and rainy periods. The mares remaining cyclically active throughout the year provided there were appropriate forage availability and quality levels to allow for normal values of body weight and condition.

The ovarian and uterine responses of Baixadeiro mares to prostaglandin synchronization during the dry and rainy seasons

This study aimed to evaluate the effect of synchronization with prostaglandin F2α in Baixadeiro mares during the rainy and dry seasons. Fourteen mares were synchronized by administering two doses of 1 mL prostaglandin PGF 2α and monitored by rectal palpation and ultrasound for the assessment of follicular development and uterine echotexture. Of this total, nine mares allowed the collection of blood, in which the blood was collected by venipuncture of the jugular vein to determine progesterone (P4) by ELISA. Mares showed no differences (P > 0.05) in weight, body score condition (BSC), tone, uterine edema, frequency of ovulation, synchronization interval, estrus, and the total number of follicles between periods. However, there was a difference in large increased follicle diameter (P < 0.05) during the dry season. The average concentrations of P4 in mares differed (P < 0.05) between the pre- and post-ovulatory phases for both seasons and after ovulation, with higher concentrations in the rainy season. Furthermore, statistical differences in daily light (P < 0.05) were observed between the dry and rainy periods. Thus, we conclude that mares from the genetic grouping Baixadeiro showed no reproductive seasonality, though there was a difference in luminosity between the rainy and dry seasons. The treatment with two doses of PGF 2α was effective in synchronizing the mares, promoting the return of estrus in the dry and rainy periods. The mares remaining cyclically active throughout the year provided there were appropriate forage availability and quality levels to allow for normal values of body weight and condition.(AU)

Níveis de progesterona e os parâmetros reprodutivos no período periovulatório de vacas Nelore submetidas ao protocolo de IATF / Progesterone levels and reproductive parameters in the periovulatory period of Nellore cows under FTAI protocol

Background: Fixed Time Artificial Insemination (FTAI) has achieved a significant evolution in the last 18 years, however, despite the progress achieved by modern FTAI programs, the conception rates obtained are still low. Therefore, this study aimed to evaluate the interrelation between progesterone levels in the periovulatory period and reproductive parameters of Nellore cows submitted to an FTAI protocol. Materials, Methods & Results: On a random day, called day 0 (D0), 57 cows received a P4 device associated with the intramuscular (IM) application of 2.0 mg of estradiol benzoate. On D9, the P4 devices were removed and then were administered 500 µg of cloprostenol sodium IM; 0.6 mg of estradiol cypionate IM and 300 IUI of Equine Chorionic Gonadotrophin IM. Blood samples were collected for the determination of serum P4 concentrations on D9 and D11 of the protocol. The evaluations of follicular diameter (DFOL), follicular wall area (AFOL) and the vascularization area of the follicle wall (VFOL) were carried out on D11 using B-mode ultrasonography examination and colour Doppler, and then the artificial inseminations were performed. The evaluation of the corpus luteum diameter (CLD), of the total corpus luteum area (CLA), of the area of corpus luteum vascularization (CLV) and blood sampling for determination of postovulatory P4 levels (Post-P4) were performed on D24. For the analysis of the P4 concentration the chemiluminescence method was used, with a sensitivity of 0.1 ng/mL. According to the P4 concentrations on D11, cows were divided into 2 groups, LOW LEVELS OF P4 and HIGH LEVELS OF P4. The diagnosis of pregnancy was performed using transrectal ultrasonography on D45, at this point the cows were divided into 2 groups, PREGNANT and NON-PREGNANT. The correlation between DFOL and P4 dosage on D11 was moderate, negative and significant and between the AFOL and the serum P4 levels on D9, was moderate, negative and significant. As for the other correlations between follicular and luteal parameters and serum P4 levels, these were low to moderate, negative and not significant. Cows in the LOW LEVELS OF P4 group had significantly larger diameter and follicular areas than the cows in the HIGH LEVELS OF P4 group, the other follicular and luteal parameters showed no statistical difference. Of the total 57 cows that were inseminated, 30 cows became pregnant. Cows in the PREGNANT group had serum P4 levels on D9 equivalent to that obtained by the NON-PREGNANT group. However, at D11 the cows that became pregnant presented significantly lower serum P4 levels than cows that did not become pregnant. Discussion: The results of the interrelation between follicular parameters and P4 levels obtained in the present study, pointed out that the lower the levels of P4, the higher the follicular parameters, corroborating with other authors. Thus, larger preovulatory follicles provided high ovulation rates. Periovulatory serum P4 levels did not significantly affect the morphofunctional parameters of the CL. Such findings may be justified by high periovulatory P4 levels resulting from less efficient luteolysis, exert a negative effect on the results of FTAI protocols, because progesterone inhibits the release of LH pulses. It is concluded that lower periovulatory P4 levels established a favourable condition for follicular development and fertility, however, morphofunctional parameters of the corpus luteum were not affected.

Abordagens genéticas e de bioengenharia para o estudo da foliculogênese / Genetic and bioengineering approaches to the study of folliculogenesis

O desenvolvimento de estudos genéticos e de microdispositivos biológicos tem proporcionado a ampliação do conhecimento sobre os complexos eventos que envolvem a reprodução animal. O desafio ainda é imensurável, mas a criação e surgimentos de novas perspectivas para a pesquisa básica tem-se feito presente. Neste trabalho revisamos de maneira suscinta algumas abordagens recentes, utilizadas pela pesquisa básica, sobretudo com o objetivo de lançar luz sobre o desenvolvimento folicular e oocitário. Dessa forma, essa revisão pretende fornecer uma visão geral do uso das tecnologias ômicas e sistema de microfluídica como auxiliadores na compreensão da foliculogênese. Adicionalmente serão apresentadas particularidades inerentes à fisiologia da gametogênese, que incluem ação de microorganismos e mitocôndrias, além do importante papel da comunicação intercelular através das vesículas extracelulares.

The development of genetic studies and biological microdevices has expanded knowledge about the complex events involving animal reproduction. The challenge is still immeasurable, but the creation and emergence of new perspectives for basic research have been present. This paper briefly reviews some recent approaches used in basic research, mainly to shed light on follicular and oocyte development. Thus, this review intends to provide an overview of the use of omics technologies and microfluidics systems as aids in understanding folliculogenesis. Also, it will present particulars inherent in the physiology of gametogenesis, which include microorganisms and mitochondria, in addition to the important role of intercellular communication through extracellular vesicles.

Histomorphometric analysis of gonads of green turtles (chelonia mydas) stranded on the coast of Espírito Santo state, Brazil / Análise histomorfométrica das gônadas de tartarugas verdes (Chelonia Mydas) provenientes de encalhe no litoral do Espírito Santo, Brasil

Studies on reproduction in sea turtles are important due to its life cycle, migratory patterns, high juvenile mortality and environmental impacts. This study aimed to analyse histomorphometrically gonads of C. mydas from the coastline of the Espírito Santo State, Brazil. Ovaries and testicles were collected between 2014 and 2015 from stranded animals. The material was fixed in formalin 10%, assessed macroscopically and processed for histomorphometrical evaluation. Gonads from 34 individuals were evaluated, twenty-four females and ten males. Macroscopic sexual identification presented 100% accuracy confirmed by histology. Sexual dimorphism was observed in one individual, which was considered as adult (CCL=1.023 m). Microscopy of female gonads revealed predominant previtellogenic follicles; oocyte diameter ranged between 161µm and 750µm and a positive correlation between ovarian length, largest oocyte and CCL was found. In males, autolysis was verified in five individuals. Viable testicles revealed predominant spermatogonia, primary spermatocytes and Sertoli cells in the seminiferous tubules and, Leydig cells and fibroblasts in the stroma. There was a positive correlation between tubular diameter and CCL and testicle length and CCL. Maturation of stromal tissue and a positive correlation between tubular lumen and CCL were also observed. Gonad development is proportional to individual growth.(AU)

Estudos em reprodução de tartarugas marinhas são importantes devido ao ciclo de vida, ao padrão migratório, à alta mortalidade juvenil e aos impactos ambientais. Objetivou-se analisar histomorfometricamente gônadas de C. mydas no litoral do Espírito Santo. Foram coletados ovários e testículos dessa espécie, entre 2014 e 2015. O material foi fixado em formol a 10% e avaliado macroscopicamente. Em seguida, foi processado para avaliação histomorfométrica. Foram avaliadas gônadas de 34 indivíduos, 24 fêmeas e 10 machos. Verificaram-se 100% de acurácia na identificação sexual à macroscopia, confirmada pela histologia. Observou-se dimorfismo sexual em um macho, que foi considerado adulto (CCC=1,023m). A microscopia dos ovários revelou folículos pré-vitelogênicos, cujos ovócitos apresentaram diâmetro médio entre 161µm e 750µm. Houve correlação positiva entre comprimento ovariano e diâmetro do maior ovócito e CCC. Nos machos, verificou-se autólise em cinco indivíduos. Os testículos viáveis revelaram espermatogônias, espermatócitos primários e células de Sertoli nos túbulos seminíferos, além de células de Leydig e fibroblastos no estroma. Houve correlação positiva entre diâmetro tubular e CCC e comprimento testicular e CCC. Verificou-se maturação do tecido estromal e correlação positiva entre o diâmetro do lúmen tubular e o CCC. Verifica-se que o desenvolvimento das gônadas é proporcional ao crescimento dos indivíduos.(AU)

Histomorphometric analysis of gonads of green turtles (chelonia mydas) stranded on the coast of Espírito Santo state, Brazil / Análise histomorfométrica das gônadas de tartarugas verdes (Chelonia Mydas) provenientes de encalhe no litoral do Espírito Santo, Brasil

Studies on reproduction in sea turtles are important due to its life cycle, migratory patterns, high juvenile mortality and environmental impacts. This study aimed to analyse histomorphometrically gonads of C. mydas from the coastline of the Espírito Santo State, Brazil. Ovaries and testicles were collected between 2014 and 2015 from stranded animals. The material was fixed in formalin 10%, assessed macroscopically and processed for histomorphometrical evaluation. Gonads from 34 individuals were evaluated, twenty-four females and ten males. Macroscopic sexual identification presented 100% accuracy confirmed by histology. Sexual dimorphism was observed in one individual, which was considered as adult (CCL=1.023 m). Microscopy of female gonads revealed predominant previtellogenic follicles; oocyte diameter ranged between 161µm and 750µm and a positive correlation between ovarian length, largest oocyte and CCL was found. In males, autolysis was verified in five individuals. Viable testicles revealed predominant spermatogonia, primary spermatocytes and Sertoli cells in the seminiferous tubules and, Leydig cells and fibroblasts in the stroma. There was a positive correlation between tubular diameter and CCL and testicle length and CCL. Maturation of stromal tissue and a positive correlation between tubular lumen and CCL were also observed. Gonad development is proportional to individual growth.(AU)

Estudos em reprodução de tartarugas marinhas são importantes devido ao ciclo de vida, ao padrão migratório, à alta mortalidade juvenil e aos impactos ambientais. Objetivou-se analisar histomorfometricamente gônadas de C. mydas no litoral do Espírito Santo. Foram coletados ovários e testículos dessa espécie, entre 2014 e 2015. O material foi fixado em formol a 10% e avaliado macroscopicamente. Em seguida, foi processado para avaliação histomorfométrica. Foram avaliadas gônadas de 34 indivíduos, 24 fêmeas e 10 machos. Verificaram-se 100% de acurácia na identificação sexual à macroscopia, confirmada pela histologia. Observou-se dimorfismo sexual em um macho, que foi considerado adulto (CCC=1,023m). A microscopia dos ovários revelou folículos pré-vitelogênicos, cujos ovócitos apresentaram diâmetro médio entre 161µm e 750µm. Houve correlação positiva entre comprimento ovariano e diâmetro do maior ovócito e CCC. Nos machos, verificou-se autólise em cinco indivíduos. Os testículos viáveis revelaram espermatogônias, espermatócitos primários e células de Sertoli nos túbulos seminíferos, além de células de Leydig e fibroblastos no estroma. Houve correlação positiva entre diâmetro tubular e CCC e comprimento testicular e CCC. Verificou-se maturação do tecido estromal e correlação positiva entre o diâmetro do lúmen tubular e o CCC. Verifica-se que o desenvolvimento das gônadas é proporcional ao crescimento dos indivíduos.(AU)

Cell proliferation in ovarian follicles fromBos taurus indicus females with different antral follicle count

The aim of this study was to compare the cell proliferation rate of follicles from Bostaurus indicusfemales with different antral follicle counts (AFCs). Thirty pairs of ovaries were classified as having low(≤ 31 follicles), intermediate (≥ 46 and ≤ 76 follicles) or high AFC (≥ 91 follicles). The ovaries were cut into 1 x 1 x 0.3 cm fragments, fixed in 10% buffered formalin solution and subjected to immunohistochemical analysis for proliferating cell nuclear antigen (PCNA). PCNA detection was detected in all stages of follicular development, being 1,388 (82.23%) primordial, 197 (11.67%) primary, 29 (1.72%) secondary and 74 (4.83%) antral follicles. The PCNA detection rate was higher (P ≤ 0.05) for antral and secondary follicles in ovaries with intermediate and low AFCs. This study showed that higher PCNA at early stages of follicular growth is detected in animals with high AFC, whereas a higher PCNA is seen in secondary and antral follicles of animals with low AFC.

Cell proliferation in ovarian follicles fromBos taurus indicus females with different antral follicle count

The aim of this study was to compare the cell proliferation rate of follicles from Bostaurus indicusfemales with different antral follicle counts (AFCs). Thirty pairs of ovaries were classified as having low(≤ 31 follicles), intermediate (≥ 46 and ≤ 76 follicles) or high AFC (≥ 91 follicles). The ovaries were cut into 1 x 1 x 0.3 cm fragments, fixed in 10% buffered formalin solution and subjected to immunohistochemical analysis for proliferating cell nuclear antigen (PCNA). PCNA detection was detected in all stages of follicular development, being 1,388 (82.23%) primordial, 197 (11.67%) primary, 29 (1.72%) secondary and 74 (4.83%) antral follicles. The PCNA detection rate was higher (P ≤ 0.05) for antral and secondary follicles in ovaries with intermediate and low AFCs. This study showed that higher PCNA at early stages of follicular growth is detected in animals with high AFC, whereas a higher PCNA is seen in secondary and antral follicles of animals with low AFC.(AU)

Resumption of cyclic ovarian activity in postpartum ewes: a review / Retorno da atividade ovariana cíclica pós-parto de ovelhas: uma revisão

The reproduction rate is considered a crucial factor that affects the ability of producers to meet the growing demand for ovines meat, and optimizing this factor will maximize the production rate and increase the efficiency of livestock. A reduction in the length of the postpartum anestrus period results in a shorter interval between lambing and conception and enables more lambs to be produced throughout the life of a ewe. This review discusses the current knowledge of several factors associated with the resumption of cyclic ovarian function in postpartum ewes and how the suckling management of ewes may affect this process. Factors that influence the resumption of cyclic ovarian activity include uterine involution, follicular development, occurrence of silent ovulation and short estrous cycles, breed, reproductive seasonality, nutrition and lactation. Controlled suckling and early weaning are management practices that may be used to obtain three lambings in a 24-month period. In the majority of the studies the interval between lambing and first estrus was reduced when a controlled suckling management strategy was used, therefore the controlled suckling appears to be the best alternative because it has produced good results and is easy to implement. Currently, however, this practice is still not widely used.(AU)

A taxa de reprodução é considerado um fator crucial que afeta a capacidade dos produtores em atender a crescente demanda por carne ovina e otimizar esse fator pode maximizar a taxa de produção e aumentar a eficiência da pecuária. A redução da duração do período de anestro pós-parto resulta em um intervalo menor entre a parição e concepção e permite que mais cordeiros sejam produzidos durante toda a vida de uma ovelha. Esta revisão discute o conhecimento atual de vários fatores associados com o retorno da função ovariana cíclica pós-parto em ovelhas e como o regime de amamentação de ovelhas podem afetar esse processo. Fatores que influenciam o retomada da atividade ovariana cíclica incluem involução uterina, o desenvolvimento folicular, a ocorrência de ovulação silênciosa e ciclos estrais curtos, raça, sazonalidade reprodutiva, nutrição e lactação. A amamentação controlada e o desmame precoce são práticas de manejo que podem ser utilizados para se obter três partos em um período de 24 meses. Na maioria dos estudos, o intervalo entre o parto e o primeiro estro foi reduzido quando o manejo de amamentação controlada foi utilizado, portanto, a amamentação controlada parece ser a melhor alternativa, pois tem apresentado bons resultados e sua implementação é fácil. Atualmente, no entanto, essa prática ainda não é amplamente utilizada.(AU)

EFEITO DA MICOTOXINA ZEARALENONA ASSOCIADA OU NÃO COM O METABÓLITO ENTEROLACTONA NO CULTIVO IN VITRO DE TECIDO OVARIANO OVINO

Os grãos produzidos na agricultura fazem parte da alimentação de humanos e animais, podendo conter fitoestrógenos, bem como contaminantes como as micotoxinas. Dentre os fitoestrogenos e as micotoxinas estrogênicas, destacam-se as lignanas e seu metabólito, a enterolactona (ENL) e a zearalenona (ZEN), respectivamente. Essas substâncias apresentam atividades estrogênicas e podem ter efeitos sobre a reprodução, não devendo negligenciar seus efeitos isolados e em associação sobre a fisiologia reprodutiva. Dessa forma, o objetivo deste estudo foi avaliar o efeito da ENL e da ZEN isoladamente ou em associação sobre a sobrevivência e a morfologia de folículos pré-antrais ovino inclusos em fragmentos ovarianos cultivados in vitro, bem como a expressão gênica e o estresse oxidativo. Ovários de 10 ovelhas foram coletados em abatedouro local e fragmentados. Os fragmentos selecionados foram distribuídos e cultivados in vitro por 3 dias a 38 °C, 5% CO2, nos seguintes tratamentos: 1- controle fresco, 2- controle cultivado, 3- ZEN (1 mol/L), 4- ENL (1 mol/L), 5- ZEN (1 mol/L) + ENL (1 mol/L). Após o cultivo in vitro, fragmentos ovarianos de todos os tratamentos foram submetidos às análises histológicas e à expressão relativa do mRNA para ABCG2, ERP29, BAX e BCL2. Os meios de cultivo foram analisados separadamente para estresse oxidativo (nitrito e malonaldeído) e níveis de estradiol. Os resultados mostraram que o cultivo in vitro reduziu as porcentagens de folículos primordiais, primários e secundários morfologicamente normais quando comparados ao controle não cultivado, exceto para folículos secundários cultivados na presença de ENL (P < 0,05). Além disso, o cultivo in vitro aumentou a densidade dos folículos primários, principalmente quando o meio de cultivo foi suplementado com ENL (P < 0,05). A adição de ENL isoladamente ou em associação com ZEN aumentou a expressão do RNAm de ABCG2 (P < 0,05). A presença dos xenoestrogênios no meio de cultivo aumentou a expressão relativa para BAX (P < 0,05). Quando comparado ao controle apenas ZEN induziu aumento na expressão de BCL2 (P < 0,05). Em conclusão, a ENL neutralizou com sucesso os efeitos deletérios da ZEN sobre a sobrevivência de folículos primordiais e primários. Entretanto a adição de ENL isoladamente não afetou a sobrevivência de folículos primordiais e primários, porém melhorou a sobrevivência de folículos secundários.

Grains produced in agriculture are part of the diet of humans and animals and may contain phytoestrogens, as well as contaminants such as mycotoxins. Among phytoestrogens and estrogenic mycotoxins, lignans and their metabolite enterolactone (ENL) and zearalenone (ZEN) stand out, respectively. These substances have estrogenic activities and may have effects on reproduction, and their isolated and associated effects on reproductive physiology should not be neglected. Therefore, the aim of this study was to evaluate the effect of enterolactone (ENL) and zearalenone (ZEN), alone or in combination, in the in vitro culture of preantral follicles enclosed in sheep ovarian tissue fragments on the survival and morphology, as well gene expression and oxidative stress. Ovaries from 10 sheep were collected at a local abattoir and fragmented. The fragments selected were distributed and cultured in vitro for 3 days at 38 °C, 5% CO2, in the following treatments: 1- fresh control, 2- cultured control, 3- ZEN (1 mol/L), 4- ENL (1 mol/L), 5- ZEN (1 mol/L) + ENL (1 mol/L). After in vitro culture, ovarian fragments of all treatments were submitted to histological analysis and the relative mRNA expression for ABCG2, ERP29, BAX, and BCL2. The culture media were separately analysed for oxidative stress (nitrite and malondialdehyde) and estradiol levels. The results showed that the in vitro culture decreased the percentages of morphologically normal primordial, primary and secondary follicles when compared to the non-cultured control, except for secondary follicles cultured in the presence of ENL (P < 0.05). Moreover, in vitro culture increased the density of primary follicles, especially when the culture medium was supplemented with ENL (P < 0.05). Addition of ENL alone or in association with ZEN increased ABCG2 mRNA expression (P < 0.05). The presence of xenoestrogens in the culture medium increased the relative expression of BAX (P < 0.05). When compared to the control, only ZEN induced an increase in BCL2 expression (P < 0.05). In conclusion, ENL successfully neutralized the deleterious effects of ZEN on the survival of primordial and primary follicles. However, the addition of ENL alone did not affect the survival of primordial and primary follicles, but it improved the survival of secondary follicles.

In vitro and in vivo embryo production in cattle superstimulated with FSH for 7 days

Over the past two decades, research efforts have resulted in superstimulation protocols that are user-friendly, but embryo production has increased only marginally. Studies to-date have not adequately answered the question of whether superstimulatory protocols can be used to overcome the follicle wave pattern, increase the number of follicles that enter the wave, or rescue a greater number of small follicles within the wave. Studies which appear to facilitate greater utilization of follicles within the wave are described in this review. The number of large follicles at the time of first AI tended to be greater, and more ovulations and CL occurred with lengthened protocol (7-day) than with the convention 4-day FSH treatment. In addition, there was greater synchrony of ovulations and the mean numbers of total ova/embryos, fertilized ova, transferable or freezable embryos were numerically higher in the 7-day group. When used in an in vitro fertilization model, FSH treatment for 7 days resulted in a greater number of follicles for aspiration, a greater proportion of expanded cumulus-oocyte-complexes, and more transferable embryos after in vitro culture. Daily ultrasonography revealed a reduction in the number of small (1-2 mm) antral follicles from the beginning to the end of the superstimulatory treatment that was associated with a progressive shift of follicles to the next size category in both 4-day and 7-day groups. The number of follicles<5 mm decreased during superstimulation suggesting that there was no continuous recruitment of small follicles, and the number of follicles>1 mm at the end of superstimulation did not differ from the number of follicles >1 mm at the beginning of Super stimulation. However, the total number of follicles >3 mm at the end of superstimulation, was greater than the number of follicles >3 mm at the beginning of superstimulation due to growth of the 1-2 mm population into larger size categories during treatment. (AU)

In vitro and in vivo embryo production in cattle superstimulated with FSH for 7 days

Over the past two decades, research efforts have resulted in superstimulation protocols that are user-friendly, but embryo production has increased only marginally. Studies to-date have not adequately answered the question of whether superstimulatory protocols can be used to overcome the follicle wave pattern, increase the number of follicles that enter the wave, or rescue a greater number of small follicles within the wave. Studies which appear to facilitate greater utilization of follicles within the wave are described in this review. The number of large follicles at the time of first AI tended to be greater, and more ovulations and CL occurred with lengthened protocol (7-day) than with the convention 4-day FSH treatment. In addition, there was greater synchrony of ovulations and the mean numbers of total ova/embryos, fertilized ova, transferable or freezable embryos were numerically higher in the 7-day group. When used in an in vitro fertilization model, FSH treatment for 7 days resulted in a greater number of follicles for aspiration, a greater proportion of expanded cumulus-oocyte-complexes, and more transferable embryos after in vitro culture. Daily ultrasonography revealed a reduction in the number of small (1-2 mm) antral follicles from the beginning to the end of the superstimulatory treatment that was associated with a progressive shift of follicles to the next size category in both 4-day and 7-day groups. The number of follicles1 mm at the end of superstimulation did not differ from the number of follicles >1 mm at the beginning of Super stimulation. However, the total number of follicles >3 mm at the end of superstimulation, was greater than the number of follicles >3 mm at the beginning of superstimulation due to growth of the 1-2 mm population into larger size categories during treatment.

PERFIL METABÓLICO E DINÂMICA FOLICULAR DE CORDEIRAS SANTA INÊS COM CONSUMO AD LIBITUM OU RESTRITO DE DIETA COM INCLUSÃO DE SOJA GRÃO TOSTADA

A restrição alimentar pode comprometer o desenvolvimento folicular ovariano. A suplementação lipídica além de aumentar o conteúdo energético da dieta, atua positivamente na dinâmica folicular e pode ser uma estratégia para reduzir possíveis efeitos prejudiciais da restrição alimentar na atividade ovariana. Os objetivos desse estudo foram avaliar o efeito da suplementação lipídica na forma de soja grão tostada e quebrada e da restrição alimentar moderada sobre a concentração sanguínea de ácido linoleico, no perfil metabólico e no desenvolvimento folicular ovariano de cordeiras Santa Inês que ovularam ou não antes da avaliação da dinâmica folicular. As hipóteses testadas foram: 1) cordeiras que receberam suplementação lipídica com soja grão tostada têm maior concentração de ácido linoleico no sangue; 2) a suplementação lipídica relaciona-se positivamente ao desenvolvimento folicular ovariano 3) a restrição alimentar moderada compromete o desenvolvimento folicular; 4) a suplementação lipídica com soja grão tostada reduz os efeitos prejudiciais da restrição alimentar moderada no desenvolvimento folicular e 5) as cordeiras que ovularam durante o período experimental tiveram melhor perfil metabólico e melhor desenvolvimento folicular do que aquelas que não ovularam. Trinta e cinco cordeiras Santa Inês com 21,5 ± 1,7 kg de peso corporal e 95,0 ± 7,9 dias de idade foram aleatoriamente distribuídas em delineamento em blocos desbalanceados, com oito blocos com quatro animais e um bloco com três animais. Os tratamentos foram organizados em um fatorial 2 x 2 x 2, onde o fator A foi o consumo (ad-libitum ou restrito), o fator B a suplementação lipídica (sem ou com suplementação lipídica com soja grão tostada) e o fator C o status fisiológico (ausência ou ocorrência de ovulação). As dietas foram fornecidas até 37,4 ± 2,95 kg. A restrição alimentar imposta foi de 15% do consumo ad-libitum. A ocorrência da ovulação foi determinada pela concentração sérica de progesterona maior ou igual a 1 ng/mL. O perfil metabólico foi avaliado aos 29,9 ± 0,56 e 35,0 ± 0,39 kg de peso corporal. A dinâmica folicular foi avaliada aos 30,7 ± 1,17 e 36,0 ± 0,91 kg de peso corporal por 17 ± 5 dias consecutivos em cada ocasião. A concentração de ácido linoleico e esteárico no sangue foi determinada aos >26-28, >28-32, >32-34, >34-38 kg de peso corporal nos animais alimentados ad-libitum e naqueles sob restrição que consumiram soja tostada. Na análise de consumo de nutrientes, ganho de peso médio diário, conversão alimentar, metabólitos sanguíneos, insulina, número de ondas, número de folículos, diâmetro do maior e do segundo maior folículo e taxa de crescimento do maior folículo foram considerados os efeitos de consumo (ad-libitum ou restrito), suplementação lipídica e status fisiológico e as respectivas interações duplas e a tripla. Na análise de ácido linoleico e esteárico foi considerado o efeito de tratamento. O consumo de matéria seca foi maior (P < 0,05) nos animais com consumo ad-libitum sem suplementação lipídica que ovularam; enquanto foi menor nos animais com consumo ad-libitum com suplementação lipídica que ovularam (interação consumo x suplementação lipidica x status fisiológico, P < 0,05). Considerando os animais alimentados ad-libitum e que receberam suplementação lipídica aqueles que não ovularam tiveram maior consumo de extrato etéreo (P < 0,05); enquanto aqueles que ovularam tiveram menor consumo de extrato etéreo (interação consumo x suplementação lipidica x status fisiológico, P < 0,05). O ganho de peso médio diário foi menor (P < 0,05) nos animais de consumo ad-libitum com suplementação lipídica e que ovularam em comparação aos que não ovularam. A conversão alimentar foi menor (P < 0,05) nos animais com consumo restrito do que naqueles com consumo ad-libitum. As concentrações glicose e de triglicerídeos foram maiores (P > 0,05) nos animais que receberam suplementação lipídica; enquanto que nenhuma alteração foi observada na concentração de insulina (P > 0,05). As concentrações séricas de HDL e colesterol foram maiores (P < 0,05) nos animais que não receberam suplementação lipídica e com consumo restrito em comparação aos animais com consumo adlibitum; enquanto não houve diferença entre os animais que receberam suplementação lipídica (interação consumo x suplementação lipidica, P < 0,05). A concentração de HDL foi maior (P < 0,05) nos animais que ovularam em relação aos que não ovularam. A concentração de colesterol não diferiu (P > 0,05) de acordo com o status fisiológico. As concentrações séricas de ácido linoléico e esteárico não foram alteradas (P > 0,05) pelo consumo ou pela suplementação lipídica. O número de ondas foi maior (P < 0,05) nos animais com consumo ad-libitum do que nos animais de consumo restrito. Os efeitos principais de tratamento, bem como as interações dupla e tripla não foram significativos (P > 0,05) no número de folículos, diâmetro do maior e do segundo maior folículos e taxa de crescimento do maior folículo. A suplementação lipídica com soja grão tostada melhorou o status metabólico, sem efeitos concomitantes na concentração de ácido linoleico no sangue e na dinâmica folicular. Exceto pela redução no número de ondas, a dinâmica folicular não foi comprometida pela restrição alimentar moderada. Apesar da maior concentração de HDL no sangue das cordeiras que ovularam, esses animais não apresentaram melhora no desenvolvimento folicular

Feed restriction can compromise ovarian follicular development. Lipid supplementation, increases the energy content of the diet and acts positively on follicular dynamics and may be a strategy to reduce possible harmful effects of feed restriction on ovarian activity. The objectives of this study were to evaluate feed restriction and lipid supplementation as roasted whole soybeans on serum concentrations of linoleic acid, on metabolic profile and on follicular dynamics of Santa Ines ewe lambs that ovulated or not before ovarian assessments. Thus, we hypothesized that 1) ewe lambs fed with roasted soybean had greater concentration of linoleic acid in the blood, 2) lipid supplementation is positively related to follicular dynamics, 3) feed restriction compromises follicular development, 4) lipid supplementation with roasted whole soybeans reduces the harmful effects of feed restriction on follicular development and 5) ewe lambs having ovulation during the experimental period had better metabolic profile and enhanced follicular development than those no having ovulation. Thirty-five Santa Ines ewe lambs with 21.5 ± 1.7 kg of body weight and 95.0 ± 7.9 days of age, were randomly distributed in unbalanced block design with eight blocks with four animals and one block with three animals. Treatments were arranged in a factorial 2 x 2 x 2 based on level of intake (ad-libitum AL or restricted FR), on lipid supplementation (without or with lipid supplementation with roasted whole soybeans) and on the physiological status (absence or occurrence of ovulation). Diets were supplied to 37.4 ± 2.95 ± kg (mean ± standard deviation). The feed restriction was 15% of ad-libitum intake. Ovulation was detected by a serum progesterone concentration 1 ng/mL. The metabolic profile was assessed at 29.9 ± 0.56 and 35 ± 0.39 kg of body weight. Follicular dynamics were assessed at 30.7 ± 1.17 and 36.0 ± 0.91 kg of body weight at 17 ± 5 consecutive days in each occasion. The serum linoleic acid concentration was determined at 26-28, 28-32, 32-34 and 34-38 kg of body weight in AL-fed animals fed and FR-fed animals that consumed roasted whole soybeans. In the analyze of nutrients intake, average daily weight gain, feed conversion, blood metabolites, insulin, number of waves, number of follicles, diameter of the largest and second largest follicle and growth rate of the largest follicle, the intake level (ad-libitum or restricted) were considered, lipid supplementation and physiological status and the respective two- and three-way interactions. For the linoleic and stearic acid serum concentration, the treatment effect were considered. Dry matter intake was greater (P < 0.05) in unsupplemented-AL animals that ovulated compared to those notovulated; conversely, dry matter intake was lower in supplemented-AL animals that ovulated compared to those not-ovulated (three-way interaction, P < 0.05). Considering that supplemented-AL animals, those that not-ovulated had a greater ether extract intake (P < 0.05); while those ovulated had ether extract intake (intake level x lipid supplementation x physiological status interaction, P < 0.05). The average daily weight gain was lower (P < 0.05) in supplemented-AL animals that ovulated compared to those that not-ovulated. Feed conversion was lower (P < 0.05) in FR-fed than AL-fed animals. Glucose and triglyceride concentrations were higher (P > 0.05) in supplemented animals; while no changes were observed in the insulin concentration (P > 0.05). HDL and cholesterol serum concentrations were higher (P < 0.05) in unsupplemented-FR compared to AL-fed animals; while there was no difference between animals that received lipid supplementation (intake level x lipid supplementation interaction, P < 0.05). The HDL concentration was higher (P < 0.05) in the animals that ovulated compared to not-ovulated animals. Cholesterol concentration did not differ (P > 0.05) according to physiological status. Linoleic and stearic acid serum concentrations were not altered (P > 0.05) by intake level or lipid supplementation. The number of waves was higher (P < 0.05) in AL-fed than FR-fed animals. The main treatment effects, as well as the two- and three-way interactions were not significant (P > 0.05) for the number of follicles, diameter of the largest and second largest follicles and growth rate of the largest follicle. In conclusion, lipid supplementation with roasted whole soybeans improve the metabolic status, with no concomitant effect on linoleic acid blood concentration and on follicular dynamics. Except by reduction in number of waves, the follicular dynamics was not compromised by moderate feed restriction. Despite the higher HDL blood concentration in ovulated ewe lambs, these animals did not present enhanced follicular development.

Efeitos do propil-parabeno e butil-parabeno na atividade reprodutiva de ratas Wistar adultas

As substâncias desreguladores endócrinas de diversas origens e propriedades químicas acomentem uma diversidade de mamíferos, animais silvestres e seres humanos, que podem interagir com o sistema reprodutivo feminino e prejudicar a homeostase hormonal. Dentro dessa classificação, os parabenos podem ser utilizados como conservantes em diversos produtos comerciais. Seus efeitos se tornam cada vez mais complexos em relação à idade, história reprodutiva, ambiente endócrino da espécime no momento da exposição, como também não é sabido todos os reais mecanismos desses compostos quando associados. Sendo assim, o objetivo deste estudo foi avaliar os efeitos dos parabenos em ratas Wistar durante um período de 90 dias consecutivos de exposição. Assim, as ratas foram distribuídas em quatro grupos experimentais: controle, que recebeu somente óleo de milho como veículo, e tratados com propil e buti-parabeno nas doses de 10 mg/kg, 100 mg/kg, ou 200 mg/kg e expostas via subcutânea. Foi utilizada a técnica de citologia vaginal para investigar a ciclicidade das fêmeas, bem como a caracterização das fases do ciclo estral pelo método Shorr. Além desses parâmetros, foram analisados aspectos morfológicos de ovários e útero, quantificação das estruturas ovarianas e avaliação ultraestutural de folículos pré-ovulatórios. Foram avaliados a concentração plasmática de estrógeno, peso corporal bem como o peso dos órgãos reprodutivos. Os resultados revelaram que o método Shorr, foi eficaz para identificar e caracterizar os tipos celulares encontrados no epitélio vaginal como a identificação de cada fase do ciclo estral. Além disso, o peso absoluto e relativo dos órgãos reprodutivos não apresentou diferença entre os grupos experimentais. A análise histológica dos ovários revelou que, todos os estágios foliculares permaneceram inalterados, como também a classificação morfológica dos Complexos Cumulus oophurus coletados. Porém, houve diminuição significativa de folículos totais primordiais e primários em fêmeas que receberam 10 e 200 mg/kg. Quanto aos folículos atrésicos houve maior incidência no grupo de 100mg/Kg. A avaliação ultraestrutural de folículos pré-ovulatórios confirmou a análise morfológica, pois permaneceram saudáveis, sem alterações a nível celular. A partir da avaliação histopatológica do útero, infiltrados leucocitários não apresentaram diferença nas proporções de células mononucleadas e polimorfonucleadas, como a intensidade. Os parâmetros histomorfométricos do tecido uterino não foram alterados como também a expressão de receptores estrogênicos (RE) no tecido uterino e ovariano. A intensidade de marcação dos receptores de progesterona no útero apresentaram diminuídas nas doses de 10 e 100 mg/Kg, e no tecido ovariano apenas a dose 100 mg/Kg. A partir dos parâmetros avaliados no presente estudo, sugere-se que após a exposição prolongada dos compostos de parabeno, a funcionalidade dos tecidos reprodutivos em fêmeas adultas pode ter sido prejudicada. Apesar da morfologia e estrutura dos órgãos sexuais permanecerem inalterados, mais estudos a nível celular e molecular precisam ser realizados para melhor entendimento da atuação conjunta dessas substâncias.

The endocrine disruption substances of many origins and chemistry properties affect a diversity of mammals, wild animals and human beings. These substances can interact with the female reproductive system and damage the hormonal homeostasis. In this classification, the paraben could be used as preservatives in different commercial products. Its effects get even worst and complex with the age, reproductive history, and endocrine environment of the specie in the moment of exposure of the drug. In complement, it is not known the actual mechanisms of those compounds when they are associated. Thus, the goal of this experiment was evaluate the effects of parabens in females Wistar during 90 consecutive days of exposure. Therefore, the females were divided in four experimental groups: Control group Corn oil; and a mixture of propil and butyl paraben with 10 mg/kg, 100 mg/kg and 200 mg/kg and exposed through the subcutaneous pathway. The vaginal cytology was performed to investigate the cyclicality as well as the characterization of the estrous phases by the Shor method. Besides that, morphological aspects of the ovary and uterus, counting of ovaries structure and ultrastructural of pre-ovulatory follicle. To complete the evaluation, the plasmatic estrogen, body weight and reproductive organ weight were assessed. The Shor method was effective to characterize and identify the cells types of the vaginal epithelium and identify the estrous cycle phase. Moreover, the relative and absolute weight have shown no difference between the experimental groups. The follicle stages had no alteration, as well as the morphological evaluating of the Cumulus oophurus by the histological analyses of the ovaries. However, there was decrease of primordial and primary follicles in females treated with 10 and 200 mg/kg. The ultrastructural evaluation of the pre-ovulatory follicles has shown no difference, confirming the morphological evaluation, with no cellular level alteration. From the histopathology evaluation, there was no difference in leukocytes infiltrates have not shown difference on proportions of mononucleated and polymorphonuclear cells, such as the intensity. No differences in the uterine tissue and expression of the estrogenic receptors were found. Regarding the progesterone receptors, there was a decrease of the expression in the uterus to the 10 and 100 mg/kg treatments, and to the 100 mg/kg dosage in the ovarian tissue. From the parameters evaluated in the present study, it is suggested that after the long exposure to the parabens, the function of the reproductive systems could be damaged. Although the morphology and the structure of the reproductive systems had no alteration, more molecular and cellular studies must be performed to the completely understand the action of these substances in association.

Porcine follicular fluid concentration of free insulin-like growth factor-I collected from different diameter ovarian follicles / Concentração do fator de crescimento semelhante à insulina tipo I no fluido folicular suíno coletado a partir de folículos ovarianos de diferentes diâmetros

The study aimed to quantify the concentrations of free IGF-I in serum and fluid of ovarian follicles in pre-pubertal gilts and describe the ovarian morphology by measuring the size of the ovaries and counting the number of surface follicles. Ovaries (n=1,000) from pre-pubertal gilts were obtained immediately after slaughter. A total of 10 samplings were performed, with ovaries obtained from 50 females for each collection. The follicles situated on the surface of each ovary were classified as small (SFs, 2 to 5mm in diameter) or large (LFs 6 to 10mm in diameter) and the follicular fluid was obtained by follicle aspiration. The collection of serum samples was performed after the gilts exsanguination using sterile tubes. From the pool of serum and follicular fluid obtained from 50 females, the concentration of free IGF-I was determined in each sample using an enzyme immunoassay kit (ELISA). The description of ovarian morphometry was performed in 100 ovaries from randomly selected gilts. The larger and smaller lengths of ovaries were measured, and the total number of SFs and LFs present on the surface of each ovary were also counted. The IGF-I concentration was greater (P<0.05) in LFs (170.92±88.29 ng/mL) compared with SFs (67.39±49.90ng/mL) and serum (73.48±34.63ng/mL). The largest and smallest length of the ovaries was 26.0±3.0 and 19.0mm ±2.0mm, respectively. The number of SFs (70.86±25.76) was greater (P<0.01) than LFs (6.54±5.26). The study concluded that LFs present greater levels of IGF-I when compared with SFs and blood, which is related to increased activity of the LFs and its differentiation to ovulation. In addition, ovaries of pre-pubertal gilts have a higher number of SFs compared to LFs. Therefore, our study demonstrated unique data regarding the physiological concentration of free IGF-I in ovarian follicles, that can be used in future research to evaluate the addition of this hormone in the in vitro production media of porcine embryos with the goal to improve the technique efficiency. (AU)

Objetivou-se quantificar as concentrações do IGF-I livre no soro e no fluido de folículos ovarianos de fêmeas suínas pré-púberes e descrever a morfologia ovariana, por meio da mensuração das dimensões dos ovários e da contagem do número de folículos superficiais. Ovários (n=1.000) foram obtidos de fêmeas pré-púberes imediatamente após o abate. Foi realizado um total de 10 coletas, sendo em cada, obtidos ovários de 50 fêmeas. Os folículos localizados na superfície de cada ovário foram classificados em pequenos (FPs, 2-5mm de diâmetro) ou grandes (FGs, 6-10mm de diâmetro) e o fluido folicular foi obtido por aspiração dos folículos. A coleta do soro foi realizada após a exsanguinação das fêmeas com o uso de tubos estéreis. A partir do pool de fluido folicular e do soro obtido das 50 fêmeas, determinou-se a concentração de IGF-I livre em cada amostra por meio de kit de ensaio imunoenzimático (ELISA). A descrição da morfometria ovariana foi realizada em 100 ovários provenientes de fêmeas escolhidas aleatoriamente. Foi mensurado o comprimento maior e menor dos ovários e, também, contabilizado o número total de FPs e FGs presentes na superfície de cada ovário. A concentração de IGF-I foi superior (P<0,05) nos FGs (170,92±88,29ng/mL) em comparação com os FPs (67,39±49,90ng/mL) e o sérico (73,48±34,63ng/mL). O comprimento maior e menor dos ovários foi de 26,0±3,0mm e 19,0±2,0 mm, respectivamente. O número de FPs (70,86±25,76) foi maior (P<0,01) em comparação com os FGs (6,54±5,26). Conclui-se que FGs apresentam níveis de IGF-I superiores aos FPs, e ao sangue, sendo isso relacionado a maior atividade dos FGs e à diferenciação que os mesmos sofrem para a ovulação. Além disso, ovários de fêmeas suínas pré-púberes apresentam elevado número de FPs em comparação aos FGs. Portanto, nosso estudo demonstrou dados originais a respeito da concentração fisiológica de IGF-I livre em folículos ovarianos, que podem ser utilizados em futuras pesquisas para avaliar a adição desse hormônio nos meios de produção in vitro de embriões suínos com o objetivo de melhorar a eficiência da técnica. (AU)

EXPRESSÃO GÊNICA OVARIANA EM CATETO (Pecari tajacu Linnaeus, 1758) APÓS TRATAMENTO HORMONAL COM GONADOTROFINAS

Dentre as espécies de animais silvestres na América Latina, o cateto (Pecari tajacu, Linnaeus 1758) vem despertando interesse comercial. Consequentemente, a caça indiscriminada associada à destruição de habitats, resulta numa diminuição do número de indivíduos de vida livre. Assim, o uso de biotécnicas reprodutivas pode gerar melhorias na manutenção dessa espécie em cativeiro e aumentarar sua produtividade, reduzindo sua caça e destruição de seus habitats. O presente estudo objetivou aplicar tratamentos hormonais de sincronização de estro em fêmeas de cateto adaptados de espécies domésticas. Assim, fêmeas foram estimuladas com dois protocolos hormonais de associação de gonadotrofinas, Dose Suína (SD) e Dose Alométrica (AD), e os tratamentos foram comparados quanto ao desenvolvimento folicular ovariano e à expressão gênica dos receptores para gonadotrofinas (FSHR e LHCGR) e dos genes que codificam o receptor de fator de crescimento transformador beta-1 (TGFR-1 ou ALK-5), receptor de proteína morfogênica óssea tipo 1A (BMPR1A ou ALK-3), receptor de proteína morfogênica óssea tipo 2 (BMPR2), além dos genes de referência gliceraldeído 3-fosfato desidrogenase (GAPDH), histona 2A (H2A) e a proteína de transcrição ubiquitinamente expressa (UXT). A resposta ovariana foi analisada quanto ao número de folículos antrais totais e classificados em três categorias quanto ao tamanho: P (<3mm), M (3-5mm) e G (>5mm). A taxa de ovulação foi registrada pelo número de corpos hemorrágicos (CH). A população pré-antral foi representada por fragmentos de córtex ovariano. A expressão dos referidos genes foi analizada por qPCR nos folículos antrais P, M, e G, em córtex e em CH. A quantidade de folículos P, M ou G foi semelhante (P>0,05) entre SD e AD. Entretanto, o tratamento SD produziu folículos G significativamente maiores (P<0,05) do que P ou M. Adicionalmente, esse tratamento produziu folículos G maiores do que o protocolo AD. Todos os genes estudados apresentaram abundância similar (P>0,05) em córtex e folículos P, M e G, comparando-se os tratamentos SD com AD. Os folículos grandes expressaram menos mRNA de FSHR (P<0,05) que córtex (SD) ou folículos pequenos (AD). Em relação ao LHCGR, a categoria M mostrou maior expressão (P<0,05) do que folículos P (no grupo SD) ou do que córtex (em AD). Os genes TGFR-1, BMPR1A e BMPR2 apresentaram menor expressão (P<0,05) em folículos M, quando comparado ao P (no grupo SD) ou ao córtex (em AD). Quanto à taxa de ovulação, o número de CH não foi alterado pela dose hormonal (P>0,05), sendo 4,00±1,17 e 2,50±0,43, para SD e AD respectivamente. Além disso a expressão de LHCGR foi similar nos CH (P>0,05) produzidos pelos tratamentos. Conforme a expressão relativa dos genes em folículos e córtex, é possível concluir que FSHR, TGFR-1, BMPR1A e BMPR2 são mais ativos no início e LHCGR na fase tardia do desenvolvimento folicular. Adicionalmente, as doses mais elevadas de eCG/hCG não aumentam a taxa de ovulação e CH não apresenta aumento da expressão de LHCGR. Finalmente, estudos são necessários para entender melhor a fisiologia reprodutiva de pecaris.

Among the species of Latin America wild animals, the collared peccary (Pecari tajacu, Linnaeus 1758) has aroused commercial interest. Consequently, indiscriminate hunting associated with habitat destruction results in a decrease in the number of free-living individuals. Thus, the use of reproductive biotechniques may generate improvements in the captive management and increase its productivity, reducing hunting and destruction of this species habitats. The present study aimed to apply hormonal treatments for estrus synchronization adapted from domestic species in peccary females. Therefore, females were stimulated with two hormonal protocols of association of gonadotropins, Swine Dose (SD) and Allometric Dose (AD), and treatments were compared for ovarian follicular development and gene expression of gonadotrophin receptors (FSHR and LHCGR) and (TGFR-1 or ALK-5), bone morphogenic protein receptor type 1A (BMPR1A or ALK-3), bone morphogenic protein receptor type 2 (BMPR2), besides housekeeping genes glyceraldehyde 3-phosphate dehydrogenase (GAPDH), histone 2A (H2A) and ubiquitinously expressed transcription protein (UXT). The ovarian response was analyzed for the number of total antral follicles and classified into three size categories: S (<3mm), M (3-5mm) and L (>5mm). The ovulation rate was recorded by the number of corpora hemorrhagica (CH). The preantral population was represented by fragments of ovarian cortex. Expression of genes was analyzed by qPCR in the antral follicles S, M, and L, in cortex and CH. The amount of follicles S, M or L was similar (P>0.05) between SD and AD. However, SD treatment produced significantly larger L follicles (P<0.05) than S or M. In addition, this treatment produced larger L follicles than AD protocol. All studied genes showed similar abundance (P>0.05) in cortex and follicles S, M and L, comparing SD treatment with AD. Large follicles expressed less FSHR mRNA (P<0.05) than cortex (SD) or small follicles (AD). Concerning LHCGR, M category showed higher expression (P<0.05) than S follicles (in the SD group) or cortex (in AD). The TGFR-1, BMPR1A and BMPR2 genes presented lower expression (P<0.05) in M follicles when compared to S (in the SD group) or to cortex (in AD). Regarding the ovulation rate, CH number was not altered by hormonal dose (P>0.05), being 4.00±1.17 and 2.50±0.43, for SD and AD respectively. In addition, the expression of LHCGR was similar between CH (P>0.05) produced by the treatments. According to the relative expression of the genes in follicles and cortex, it is possible to conclude that FSHR, TGFR-1, BMPR1A and BMPR2 are more active at the beginning and LHCGR at the late phase of follicular development. Additionally, higher doses of eCG/hCG do not increase ovulation rate and LHCGR expression does not increase in CH. Finally, studies are needed to better understand the reproductive physiology of peccaries.

Retorno á ciclicidade ovariana e indução da ovulação em vacas Nelore utilizando eCG e GnRH

O objetivo geral da pesquisa foi desenvolver um protocolo baseado em eCG, GnRH e PGF2 que induza o retorno à ciclicidade de vacas Nelore em anestro. Para isso foram construídos dois experimentos. O experimento I teve como objetivos específicos determinar o intervalo de dias entre a aplicação da eCG e o momento de aquisição de capacidade ovulatória e determinar o percentual de vacas que ovularam em resposta à administração de GnRH. Já o experimento II teve como objetivos específicos determinar a taxa de ovulação após a primeira e após a segunda injeção de GnRH em vacas submetidas a um protocolo eCG-GnRH-PGFGnRH. No experimento I (n = 20), a porcentagem de vacas que apresentou folículos com capacidade ovulatória dois dias após a aplicação de 800 UI, 400 UI e 0 UI de eCG foi de 100%, 71,42% e 66,66%, respectivamente. A taxa de ovulação após uma única injeção de GnRH, foi de 100%, 85,7% e 66,66%, respectivamente e, não houve diferença em função da aplicação ou não da eCG sobre a taxa de ovulação (P = 0,17). No experimento II, as vacas (n = 21) receberam 400 UI de eCG (Dia 0), seguida por GnRH (D2), PGF2 (Dia 8) e GnRH (D10). A porcentagem de animais que respondeu à primeira injeção de GnRH foi maior que a segunda, (71,42% vs. 19,04%, P < 0,001), sendo que 23,8% dos animais não responderam a nenhuma das injeções de GnRH. Desta forma, a eCG é efetiva em induzir a emergência de uma nova onda folicular dois dias após sua aplicação em vacas Nelore em anestro e, portanto, efetiva em auxiliar o retorno à ciclicidade. O protocolo baseado no uso de eCG, GnRH e PGF2 foi eficiente em induzir a ovulação sincronizada da maior parte dos animais após a primeira injeção de GnRH, porém, não após a segunda. Desta forma, pesquisas adicionais são necessárias a fim de melhorar a eficiência deste protocolo com relação à taxa de vacas com ovulação sincronizada à segunda injeção de GnRH.

The objective of this study was to develop a protocol based on eCG, GnRH and PGF2 to induce resumption of cyclicity in anovular Nelore cows, for which two experiments were conducted. Experiment I aimed to determine the day interval between eCG injection and acquirement of ovulatory capacity and to estimate the percentage of cows that ovulated in response to GnRH injection. Experiment II was designed to determine the ovulation rate after the first and second injection of GnRH of cows that were submitted to a eCG-GnRH-PGFGnRH protocol. In Experiment 1 (n = 20), the percentage of cows that presented follicles with ovulatory capacity within two days after the administration of 800, 400, and 0 IU of eCG was 100%, 71.42%, and 66.66%, respectively. The ovulation rate after a single GnRH injection was 100%, 85.7%, and 66.66%, respectively; and there was no difference between eCG administration and none on the ovulation rate (P = 0,17). In Experiment 2, cows (n = 21) received 400 IU of eCG on Day 0, followed by GnRH (Day 2), PGF2 (Day 8) and GnRH (Day 10). The percentage of cows that responded to the first GnRH injection was higher than to the second, (71.42% vs. 19.04%, P < 0.001), considering that 23,8% of cows did not respond to any GnRH injection. Therefore, eCG is effective on inducing emergence of a new follicular wave within two days after its administration in anestrous Nelore cows; hence, it is effective to support the resumption of cyclicity. The protocols based on the administration of eCG, GnRH and PGF2 have been effective in inducing synchronized ovulation in the majority of cows after the first GnRH injection; however, not as effective after the second injection of GnRH. Consequently, additional studies are essential to enhance the efficiency of this protocol concerning the synchronized ovulation rate following the second GnRH injection.

DINÂMICA FOLICULAR OVARIANA E PADRÃO HORMONAL DAS ÉGUAS DO GRUPAMENTO GENÉTICO BAIXADEIRO

O estudo teve como objetivo avaliar a estacionalidade reprodutiva por meio do desenvolvimento folicular pré e pós-ovulatório e da concentração de progesterona nos períodos seco e chuvoso. As éguas (n=14) foram sincronizadas com duas doses intervaladas de PGF2. tiveram peso e ECC acompanhados durante o período experimental. Foram avaliadas por ultrassonografia em dias alternados para registro do desenvolvimento folicular, assim como os dados de tonicidade e morfoecogenicidade uterina. A avalição tornou-se diária quando apresentaram folículos >30mm. Para o ensaio hormonal, foram utilizadas nove éguas, coletando-se sangue em tubos heparinizados em dias alternados, com início 72h após a sincronização e estendendo-se até 10 dias após a ovulação. Dados climáticos para o período também foram analisados. Para as 14 éguas, não houve diferença estatística (p>0,05) quanto à sincronização, peso e ECC entre os períodos chuvoso e seco, assim como também não apresentaram diferença significativa na taxa de ovulação e para as características do desenvolvimento folicular, exceto para folículos médios e grandes que foram maiores no período chuvoso e seco, respectivamente. Do ensaio hormonal, houve diferença estatística para taxa de ovulação.

The study aimed to evaluate the reproductive seasonality through pre follicular development and post-ovulatory and progesterone concentration in the dry and rainy seasons. The mares (n = 14) were synchronized with two spaced doses of PGF2. had weight and BSC followed during the trial period. They were evaluated by ultrasonography every other day for registration of follicular development, as well as data and uterine tone morphoechogenicity. This evaluation became daily when presented follicles > 30mm. To test the hormonal nine mares were used, collecting blood into heparinized tubes on alternate days beginning 72h after synchronization and extending to 10 days after ovulation. Climatic data for the period were also analyzed. For the 14 mares, there was no statistical difference (p> 0,05) for synchronization, weight and BSC between the rainy and dry seasons, as well as no significant difference in ovulation rate and the characteristics of follicular development, except for medium and large follicles which were higher in the rainy season and dry, respectively. Hormone test, there was no statistical difference for ovulation rate.