Bone Morphogenetic Protein-6 (BMP-6) Stimulates the Antrum Formation by the Regulation of its Signalling Pathway in Caprine Pre-antral Follicles Cultured In Vitro.

BMP-6 has been found to be important to ovarian cells and oocyte, as well as to uterus. Thus, this study investigated the effect of bone morphogenetic protein (BMP-6) and recombinant follicle-stimulating hormone (rFSH) alone or in combination on the in vitro culture (IVC) of isolated caprine secondary follicles (Experiment 1) and the mRNA levels for BMP receptors/Smad signalling pathway (BMPR1A, BMPR2, SMAD1, SMAD4, SMAD5, SMAD6, SMAD7 and SMAD8) in vivo and in vitro using BMP-6 (Experiment 2). Secondary follicles were cultured in αMEM(+) alone (control medium) or supplemented with BMP-6 at 1 or 10 ng/ml and rFSH alone or the combination of both BMP-6 concentrations and rFSH. The results from Experiment 1 showed that the antrum formation rate was higher in the BMP-6 at 1 ng/ml (p < 0.05) than in MEM. In Experiment 2, the mRNA expression for BMPR2, SMAD1, SMAD5 and SMAD6 was detected in non-cultured control and after in vitro culture (MEM and 1 ng/ml BMP-6); while the expression of SMAD7 and SMAD8 mRNA was only detected after IVC, SMAD4 was only detected in the BMP-6 at 1 ng/ml treatment. In conclusion, the low BMP-6 concentration positively influenced antrum formation and ensured normal mRNA expression for BMP receptor and Smads after IVC of caprine secondary follicles.

Semen variables and sperm membrane protein profile of Saanen bucks (Capra hircus) in dry and rainy seasons of the northeastern Brazil (3°S).

The Saanen is a highly productive breed, and for this reason, it has been raised in Brazil, but mostly under climate conditions completely different from where the breed originated. The objective of this study was to investigate variations in semen parameters and sperm membrane proteins from Saanen bucks (n = 7) raised in Northeastern Brazil, during dry season (September, October, and November) and rainy season (March, April, and May). We showed that during the dry season, sperm motility, concentration, and the percentage of normal sperm decreased as compared to the rainy season. Rectal temperatures of bucks had no significant (p > 0.05) variations during the dry and rainy seasons. However, temperatures of left and right skin testis were higher (p < 0.05) during the dry as compared to the rainy season. Expression of three proteins (lysine-specific demethylase 5D, adenosine triphosphate (ATP) synthase subunit d, and radial spoke head protein 9 homolog) in sperm membrane were more intense in rainy season and only one protein (cytosol aminopeptidase) had greater expression in the dry season of the year. Our results show that mechanisms of testicular thermoregulation of Saanen bucks did not prevent a decrease in seminal parameters during the dry season. This deterioration may be related to reduced expression of proteins associated with important functions in sperm membrane.

Dynamic medium containing growth differentiation factor-9 and FSH maintains survival and promotes in vitro growth of caprine preantral follicles after long-term in vitro culture.

The aim of the present study was to evaluate the effects of growth differentiation factor 9 (GDF-9) and FSH on the in vitro development of caprine preantral follicles cultured for 16 days. Ovarian fragments were cultured in αMEM⁺ (α-minimum essential medium, pH 7.2-7.4, 10 µg mL⁻¹ insulin, 5.5 µg mL⁻¹ transferrin, 5.0 ng mL⁻¹ selenium, 2 mM glutamine, 2 mM hypoxanthine and 1.25 mg mL⁻¹ bovine serum albumin) in the absence or presence of 200 ng mL⁻¹ GDF-9 and/or 50 ng mL⁻¹ FSH added during the first (Days 0-8) and/or second (Days 8-16) half of the culture period. Non-cultured and cultured fragments were processed for histological and ultrastructural analyses. After 16 days, all treatments using GDF-9 or FSH showed higher rates of follicular survival compared with αMEM⁺ alone. Compared with non-cultured control, sequential culture media containing GDF-9 and/or FSH significantly increased the percentage of developing follicles and follicle diameter. Moreover, a progressive increase in oocyte diameter was observed only with sequential culture medium containing GDF-9 until Day 8 followed by FSH (GDF-9/FSH) in the second half of the culture period. After 16 days of culture, ultrastructural analysis confirmed the integrity of follicles cultured in the presence of GDF-9/FSH. In conclusion, a dynamic medium containing GDF-9 and FSH (GDF-9/FSH) maintained follicular integrity and promoted activation of primordial follicles and growth during long-term in vitro culture of goat preantral follicles.

Effect of heat stress on the survival and development of in vitro cultured bovine preantral follicles and on in vitro maturation of cumulus-oocyte complex.

The deleterious effect of heat stress (HS) on competence of oocytes from antral follicles is well recognized, but there is a lack of data regarding its impact on the viability and growth of preantral follicles. In this study, we used in vitro preantral follicle cultures to investigate the effects of HS on the following parameters: survival and development of primordial follicles after in vitro culture of ovarian fragments (experiment I); growth and antrum formation of isolated advanced secondary follicles (experiment II); and maturation rates after in vitro maturation (IVM) of cumulus-oocyte complexes (COCs) from antral follicles (>2-6 mm) grown in vivo (experiment III). Furthermore, the following end points were evaluated in all experiments: follicle/oocyte survival, reactive oxygen species (ROS), estradiol (E2) and progesterone (P4) production, as well as mRNA expression for select genes related to stress (HSP70) and apoptosis (MCL1 and BAX). In all experiments, HS consisted of exposing the structures (ovarian fragments, isolated preantral follicles and COCs) to 41 °C for 12 hours and then to 38.5 °C until the end of the culture (7 days for experiments I and II and 24 hours for experiment III). The temperature for the control group was held at 38.5 °C for the entire culture period. Heat stress increased (P < 0.05) the percentage of developing follicles (intermediate, primary, and secondary follicles) at 12 hours and increased levels of ROS at all evaluated time points (12, 24 hours, and D7), when compared to the control (experiment I). Heat stress did not affect (P > 0.05) any identified end points when preantral follicles were cultured in their isolated form (experiment II). However, in experiment III, HS decreased (P < 0.05) both the rates of metaphase II after 24 hours and E2 production at 12 hours of IVM. Moreover, HS increased (P < 0.0001) levels of P4 after IVM and ROS production at every evaluated time point, compared with the control (12 and 24 hours). In conclusion, HS caused: (1) early activation of primordial follicles; (2) an increase in ROS production by early preantral follicles enclosed in ovarian tissue and by COCs; (3) a short-term reduction of E2 production by COCs; and (4) an increase in P4 secretion from COCs. However, HS did not affect in vitro culture of advanced isolated secondary follicles. Experimental evidence indicates that preantral follicles are less sensitive to HS than COC.

Impact of growth hormone (GH) and follicle stimulating hormone (FSH) on in vitro canine preantral follicle development and estradiol production.

OBJECTIVE: Evaluate the effect of different concentrations of growth hormone (GH) on the in vitro development of domestic dog (Canis lupus familiaris) preantral follicles in the presence or absence of follicle stimulating hormone (FSH). METHODS: Secondary preantral follicles, isolated by microdissection, were cultured in a medium composed of αMEM with bovine serum albumin (BSA), glutamine, hypoxanthine, insulin, transferrin, selenium and ascorbic acid (αMEM(+)-control) added at different concentrations of GH (GH10 ng/ml or GH50 ng/ml) and FSH (GH10+FSH, GH50+FSH). Follicle development was evaluated based on the percentage of intact follicles, antrum formation, follicular diameter, follicular viability using fluorescent markers and estradiol production. RESULTS: GH50 was the only treatment that maintained the same percentage of normal morphologically follicles from day 0 to day 18 of culture (P<0.05). For all treatments, except the control, follicles were viable throughout the 18 days of culture (P<0.05). GH50 supplemented with FSH (GH50+FSH) resulted in the highest average follicular diameter (P<0.05) from day 12 to 18. Follicles from both the control and the GH50+FSH treatment groups actively and increasingly secreted estradiol from day 6 to 18 of culture (P<0.05). CONCLUSIONS: Our study demonstrates that GH benefits the maintenance of follicular morphology in a dose-dependent manner and, in association with FSH, stimulates in vitro follicular growth and estradiol production.

Differential gene expression and immunolocalization of platelet-derived growth factors and their receptors in caprine ovaries.

This study evaluated the messenger RNA (mRNA) expression and immunolocalization of all members of the platelet-derived growth factor (PDGF) family in caprine ovaries by quantitative PCR and immunohistochemistry, respectively. Detectable levels of PDGF-A mRNA were not observed in primordial follicles. Higher levels of PDGF-B mRNA were observed in primary follicles than in primordial follicles (P < 0.05). PDGF-D mRNA levels were higher in secondary follicles than in the other preantral follicle categories (P < 0.05). PDGF-B mRNA expression was higher than PDGF-C mRNA expression in primary follicles (P < 0.05). In antral follicles, PDGF-A mRNA expression was higher in cumulus-oocyte complexes (COCs) from small antral follicles than in those from large antral follicles and their respective granulosa/theca (GT) cells (P < 0.05). Furthermore, in COCs from small and large antral follicles, PDGF-A mRNA expression was higher than that of the other PDGF isoforms (P < 0.05). The mRNA levels of PDGF-B and PDGF-D and PDGFR-α and PDGFR-ß were higher in GT cells from large antral follicles than in GT cells from small antral follicles and in their respective COCs (P < 0.05). In COCs and GT cells from small antral follicles, the mRNA levels of PDGFR-α were higher than those of PDGFR-ß (P < 0.05). All proteins were observed in the cytoplasm of oocytes from all follicular categories. In granulosa cells, all PDGFs and PDGFR-ß were detected from starting at the secondary stage, and in theca cells, all proteins, except PDGF-C, were detected starting at the antral stage. In conclusion, PDGF and its receptors are differentially expressed in the oocytes and ovarian cells according to the stage of follicular development, suggesting their role in the regulation of folliculogenesis in goats.

The effects of epidermal growth factor (EGF) on the in vitro development of isolated goat secondary follicles and the relative mRNA expression of EGF, EGF-R, FSH-R and P450 aromatase in cultured follicles.

The effects of varying concentrations of EGF were evaluated in terms of in vitro follicular development and the mRNA expression levels of EGF, EGF-R, FSH-R and P450 aromatase. After 6 days, the addition of 50 ng/mL of EGF to the culture medium increased the antrum formation rates in comparison to cultured control and after 18 days of culture produced oocytes with higher rates of meiosis resumption when compared to the other treatments (P<0.05). The daily follicular growth rates in presence of EGF (50 or 100) were increased in comparison to the cultured control (P<0.05). Treatment with EGF 50 stimulated the expression of EGF mRNA but reduced EGF-R mRNA expression and estradiol secretion as compared to the cultured control (P<0.05). After 18 days of culture, the mRNA levels for FSH-R and P450 aromatase were greater than those of the non-cultured controls (P<0.05). In conclusion, the effects of EGF treatment on the mRNA levels for EGF, EGF-R, FSH-R, and P450 aromatase varied according to the stage of follicle development.

Sperm parameters and biochemical components of goat seminal plasma in the rainy and dry seasons in the Brazilian Northeast: the season's influence on the cooling of semen / Caracrterísticas espermáticas e bioquímicas do plasma seminal de caprinos nas estações chuvosa e seca do Nordeste brasileiro: influência da estação no resfriamento do sêmen

The present study aimed to verify the caprine semen characteristics during dry and rainy seasons in the Brazilian Northeast, and the influence of these seasons on cooled semen. Seminal volume, concentration, percentage of motile cells, vigor and spermatic morphology, as well as biochemical profile (fructose, citric acid, P, Ca2+, Mg, total proteins and phospholipase A2 activity) were analyzed. It was observed a reduction (P<0.05) in normal sperm morphology, fructose, citric acid, P, Mg and total protein concentration during the dry season, which did not affect the motility, vigor, volume and sperm concentration. Phospholipase A2 activity was increased during the dry season (P<0.05). The analysis of the semen cooled at 4ºC during 48 hours showed reduction in total motility and vigor sperm during the dry season (P<0.05). Based on these results, we conclude that the best period of year for caprine semen cooling is the rainy season.

Verificou-se as características seminais de caprinos durante a época seca e a chuvosa no Nordeste brasileiro e a influência da época no resfriamento do sêmen. Foram mensurados volume, concentração espermática, porcentagem de espermatozoides móveis, vigor, morfologia espermática e características bioquímicas (frutose, ácido cítrico, fósforo, magnésio, proteínas totais e atividade da fosfolipase A2). Observou-se redução (P<0,05) no número de espermatozóides morfologicamente normais, frutose, ácido cítrico, fósforo, magnésio e proteínas totais durante a época seca que não influenciaram na motilidade, vigor, volume e concentração do sêmen. Entretanto, a atividade da fosfolipase A2 foi maior na época seca. Quando o sêmen foi submetido ao resfriamento a 4ºC durante 48 horas, houve redução (P<0,05) na motilidade total e no vigor espermático durante a época seca. Com base nesses resultados, conclui-se que o período chuvoso é melhor para resfriar sêmen de caprinos no Nordeste brasileiro.

Avaliação de espermatozoides caprinos congelados em meio à base de água de coco em pó (ACP-101®) ou TRIS / Evaluation of goat spermatozoa frozen in media based on powder coconut water media based (ACP-101®) or TRIS

Compararam-se as características cinéticas e morfológicas de espermatozoides caprinos congelados nos meios à base de ACP-101® e TRIS. Os diluentes utilizados foram: ACP-101® (+ 2,5 por cento gema ovo + 7 por cento glicerol) e TRIS (+ 20 por cento gema ovo + 6,8 por cento glicerol). Quarenta e oito ejaculados de quatro bodes foram coletados, avaliados, divididos em duas alíquotas e diluídos nos meios ACP-101® e TRIS, respectivamente, posteriormente congelados e, após 30 dias, descongelados. A avaliação da motilidade espermática por computador foi realizada aos 5, 60 e 120 minutos pós-descongelação. As características de motilidade espermática analisadas foram: motilidade total (MT) ( por cento) e progressiva (MP) ( por cento), velocidades média do trajeto do espermatozoide (VAP) (µm/s) e linear (VSL) (µm/s) e população de espermatozoides rápidos (ER) ( por cento). As avaliações de morfologia espermática quantificaram a porcentagem de espermatozoides normais (N) e as alterações da cabeça (AC), da peça intermediária (API) e do flagelo (AF), aos cinco e 120 minutos pós-descongelação. O diluente TRIS apresentou resultados cinéticos mais elevados que o ACP-101® aos 60 e 120 minutos pós-descongelação. As AC aos 120 minutos pós-descongelação foram mais altas nos espermatozoides congelados em ACP-101®. Conclui-se que o diluente TRIS promoveu maior viabilidade in vitro dos espermatozoides caprinos pós-descongelação.

The aim of the work was to compare kinetic and morphologic characteristics of goat sperm frozen in diluent media based on ACP-101® and TRIS. The employed diluents were: ACP-101® (+ 2.5 percent egg yolk + 7 percent glycerol) and TRIS (+ 20 percent egg yolk + 6.8 percent glycerol). Forty eight ejaculates from four bucks were collected, assessed and divided into two aliquots and diluted into the experimental treatments ACP-101® and TRIS. The samples were frozen and after 30 days, thawed. The computer assisted spermatic motility evaluations were placed into 5, 60 and 120 minutes post-thawing. The motion parameters assessed were: total motility (MT) ( percent), progressive motility (MP) ( percent), average path velocity (VAP) (µm/s), straight linear velocity (VSL) (µm/s), and population of rapid spermatozoa (ER) ( percent). The morphologic parameters: normal spermatozoa (N), head alteration (AC), intermediary piece alteration (API), tail alteration (AF) were evaluated at 5 and 120 min post-thawing. The media based on TRIS showed kinetic results significantly superior to ACP-101® at 60 and 120min. After 120min post-thawing the AC was higher in frozen sperm in media based on ACP-101®. The TRIS media promoted better goat spermatozoa in vitro viability post-thawing.

Consumo, produção de leite e estresse térmico em vacas da raça Pardo-Suíça alimentadas com castanha de caju / Dry mater intake, milk yield, and heat stress indicators of dairy cows fed diets with cashew nut

Avaliaram-se o consumo de matéria seca, a produção de leite e os indicadores de estresse térmico de vacas Pardo-Suíça alimentadas com castanha de caju no semi-árido do Nordeste do Brasil. Doze animais foram distribuídos em um ensaio de reversão, com quatro tratamentos: 0, 8, 16 e 24 por cento de castanha no concentrado. As vacas receberam cana-de-açúcar à vontade e sete quilos de concentrado por dia. Maior consumo de matéria seca de cana-de-açúcar foi observado no tratamento com concentrado sem castanha (7,70kgMS/dia) em relação aos tratamentos com 16 por cento e 24 por cento de castanha (7,35 e 7,05kgMS/dia, respectivamente). O consumo no tratamento com concentrado sem castanha não diferiu do consumo no tratamento com 8 por cento (7,59kgMS/dia). Não houve efeito dos tratamentos sobre a produção de leite e sobre as variáveis indicativas de estresse térmico (P>0,05)

A study was carried out to evaluate dry matter intake, milk yield, and heat stress parameters in Brown Swiss cows fed diets with cashew nut. Animals were raised in the semi-arid region of the Brazilian Northeast. Twelve cows were subjected to a switch back experimental design, with four treatments: 0, 8, 16, and 24 percent of cashew nut in the concentrate. Each cow received 7kg of concentrate per day and had free access to sugar cane. Dry matter (DM) intake and milk yield were daily taken as well as measurements of rectal and milk temperature; and cardiac and respiratory rates. The highest intake of forage (sugar cane) was obtained when the concentrate had no cashew nut (7.7kgDM/day). This value was not different when the concentrate contained 8 percent of cashew nut (7.59kgDM/day) but greater than dry matter intake of cows receiving diets with 16 percent of cashew nut (7.35kgDM/day; P<0.05). The diet with 24 percent of cashew nut in the concentrate resulted in the lowest consumption of forage (7.05kgDM/day), which was significantly different from all other treatments (P<0.05). Variations in milk yield (from 14.76 to 15.31kg/day) were not related to changes in the content of cashew nut in the concentrate (P>0.05). Such low variability in daily milk yield could be associated with the higher energy density of diets containing more cashew nut. Finally, indicators of heat stress were not influenced by changes in the diets, given the air temperatures and environment where all cows were raised