Diferentes momentos de aplicação de gonadotrofina coriônica equina em protocolo de inseminação artificial em tempo fixo para vacas de leite / Different times of application of equine chorionic gonadotropin in fixed-time artificial insemination protocols for dairy cows

Avaliaram-se diferentes momentos de aplicação da gonadotrofina coriônica equina (eCG) em protocolos de inseminação artificial em tempo fixo (IATF) para vacas de leite. Foram utilizadas 76 fêmeas, as quais receberam, no dia zero (D0) do protocolo, dispositivos intravaginais de progesterona, sendo esses retirados no D9, e os animais foram, então, distribuídos aleatoriamente em três tratamentos: T1 - aplicação de eCG no momento da retirada dos dispositivos; T2 e T3 - aplicação de eCG 48h e 24h antes da retirada dos dispositivos, respectivamente. No D10 os animais receberam 1mg de GnRH, e a IATF foi realizada 52 horas após a retirada do implante. Não houve diferença (P>0,05) para intervalo entre a retirada do implante à ovulação (72,56±3,92h), o diâmetro do maior folículo no D9 (10,88±1,49mm), o diâmetro do folículo ovulatório (15,15±1,16mm) e do segundo maior folículo (7,49±0,52mm), a taxa de crescimento folicular (1,38±0,04mm/dia), a taxa de ovulação (96,67%), o intervalo entre diâmetro final e inicial do folículo dominante (73,49±3,84h), a área de corpo lúteo (2,27±0,43cm²), a porcentagem de CL no ovário direito (53,00%) e no esquerdo (26,33%) e a taxa de gestação (33,33%). O momento da aplicação da eCG não influenciou na eficiência do protocolo. Recomenda-se a utilização da eCG no momento da retirada do implante por otimização do manejo.(AU)

Different times of application of equine chorionic gonadotropin (eCG) in Fixed-Time Artificial Insemination (FTAI) protocols for dairy cows were evaluated. A total of 76 females were used, which received intravaginal progesterone devices on day zero (D0) of the protocol, which were withdrawn on D9, and the animals were then randomly distributed in three treatments: T1- application of eCG at the time of device withdrawal; T2 and T3 - application of eCG 48h and 24h before withdrawal of the devices, respectively. On D10 the animals received 1mg of GnRH and the FTAI was carried out 52 hours after the removal of the implant. There was no difference (P>0.05) for interval from implant removal to ovulation (72.56±3.92h), diameter of the largest follicle of D9 (10.88±1.49mm), diameters of ovulatory follicle (15.15±1.16mm) and of the second largest follicle (7.49±0.52mm), follicle growth rate (1.38±0.04mm/day), ovulation rate (96.67%), interval between final diameter and initial diameter of dominant follicle (73.49±3.84h), corpus luteum area (2.27±0.43cm²), percentage of CL in right ovary (53.00%) and in left ovary (26.33%) and pregnancy rate (33.33%). The time of the application of eCG did not influence the efficiency of the protocol. We recommend the use of eCG at the time of the removal of the implant for management optimization.(AU)

Farmacopuntura utilizando os acupontos Hou Hai e Bai Hui em protocolos de sincronização de estro em cabras / Pharmacopuncture using acupoints Hou Hai and Bai Hui in estrous synchronization protocols in goats

Avaliou-se a eficiência da administração de subdoses de eCG nos acupontos Bai Hui e Hou Hai em protocolos de sincronização de estro em cabras. Na primeira etapa, 57 cabras foram distribuídas aleatoriamente em quatro tratamentos: T1- 300UI de eCG intramuscular (IM); T2- 60UI de eCG no acuponto Hou Hai; T3- 60UI de eCG no acuponto Bai Hui e T4- 60UI de eCG IM; e na segunda etapa, 28 cabras foram distribuídas aleatoriamente em três tratamentos: T1- 300UI de eCG IM; T2- 30UI de eCG no acuponto Bai Hui e T3- 30UI de eCG IM. Ao final do tratamento hormonal, as cabras foram monitoradas para detecção do estro, realização das coberturas e avaliação do comportamento reprodutivo. Os dados foram submetidos à análise de normalidade, seguida dos testes estatísticos adequados para cada variável. Na primeira etapa experimental, obteve-se maior duração de estro nas cabras do T1 (P=0,009). Na segunda etapa experimental, obteve-se maior número de animais em estros no T1 (P=0,03). As demais variáveis para ambas as etapas não sofreram influência dos tratamentos (P>0,05), demonstrando que a administração de subdoses de eCG nos acupontos Bai Hui e Hou Hai foi eficiente para sincronizar o estro.(AU)

The efficiency of administration of subdoses of eCG in the Bai Hui and Hou Hai acupoints in oestrus synchronization protocols in goats was evaluated. In the first stage, 57 goats were randomly assigned to four treatments: T1- 300UI of intramuscular eCG (IM); T2- 60UI of eCG in acupoint Hou Hai; T3- 60UI of eCG in the Bai Hui acupoint and T4- 60UI of eCG IM; and in the second stage, 28 goats were randomly assigned to three treatments: T1-300UI of eCG IM; T2-30UI of eCG in the Bai Hui acupoint and T3- 30UI of eCG IM. At the end of the hormonal treatment the goats were monitored for estrus detection, and evaluation of reproductive behavior. The data were submitted to normality analysis, followed by appropriate statistical tests for each variable. In the first experimental stage, a longer duration of estrus in the T1 goats (P= 0.009) was obtained. In the second experimental stage, a greater number of animals were obtained in estrus at T1 (P= 0.03). The other variables for both experiments were not influenced by the treatments (P> 0.05), demonstrating that administration of eCG subdoses in the Bai Hui and Hou Hai acupoints was efficient to synchronize the estrus.(AU)

Eficácia da associação dupla dose PGF2 alfa-eCG no proestro de vacas leiteiras mestiças submetidas à IATF / Efficacy of the double PGF2 alpha dose-eCG association in proestrus of crossbred dairy cows submitted to IATF

Objetivou-se avaliar o efeito de uma ou duas doses de prostaglandina F2α (PGF2α) associada ou não a gonadotrofina coriônica equina (eCG) sobre a dinâmica folicular, a função luteal pré-ovulatória, assim como as características morfofuncionais pós-ovulatórias do corpo lúteo (CL) em fêmeas mestiças cíclicas submetidas a um protocolo de inseminação artificial em tempo fixo (IATF). Para tanto, 29 vacas 3/4 Gir x Holandês multíparas foram submetidas ao exame de ultrassonografia (US) transretal e após a detecção do CL iniciou-se um protocolo de IATF em um dia denominado zero (D0), por meio da inserção do implante de progesterona (P4) associado à aplicação de 2,0mg de benzoato de estradiol. No D7 esses animais receberam 12,5mg de dinoprost trometamina. No D9 realizou a remoção dos dispositivos de P4 e aplicou 0,6mg de cipionato de estradiol. Nesse momento, as fêmeas foram subdivididas nos seguintes tratamentos: Grupo Controle (n=7), foi administrado 2,5mL de solução fisiológica; Grupo 2PGF (n=7), aplicou 12,5mg de dinoprost trometamina; Grupo eCG (n=7), administrou-se 300UI de eCG; Grupo 2PGF+eCG (n=8), realizou a aplicação de 300UI de eCG e 12,5mg de dinoprost trometamina. Para avaliar a dinâmica folicular foram realizados exames de US em modo B e power doppler (Mindray Z5, Shenzhen, China) a cada 12h do D7 até o momento da ovulação ou 96h após a remoção dos implantes de P4, mensurando-se o diâmetro folicular (DFOL), a área da parede folicular (AFOL) e a área de perfusão sanguínea da parede folicular (VFOL). Concomitante a cada exame, foram coletadas amostras de sangue sendo determinada a concentração sérica de P4 pré-ovulatória por meio da metodologia de quimioluminescência. No D24 foi realizada a US modo B e doppler analisando-se o diâmetro luteal (DCL), área luteal (ACL) e área de perfusão sanguínea do CL (VCL), assim como, foi coletada amostra de sangue para averiguar a concentração sérica de P4 pós-ovulatória. Os dados foram avaliados pelo Two-way ANOVA e análise de medidas repetidas considerando os efeitos do eCG, 2PGF e interação eCG*2PGF, P<0,05. Não houve diferença significativa entre os protocolos de sincronização para as variáveis DFOL, AFOL e VFOL ao longo do tempo da dinâmica folicular. Os grupos experimentais apresentaram uma concentração sérica de P4 pré-ovulatória semelhante em cada momento da avaliação. Não foi observada distinção da ACL e VCL entre os tratamentos hormonais, contudo o Grupo eCG demonstrou tendência (P=0,08) a apresentar maior DCL em relação ao Grupo 2PGF e 2PGF+eCG. Adicionalmente a estes achados, também foi constatado tendência (P=0,07) a maiores concentrações de progesterona no dia 24 do protocolo nos animais do Grupo eCG (11,00±3,32ng/mL) em relação ao Grupo 2PGF (6,37±1,31ng/mL), enquanto o Controle e 2PGF+eCG demonstraram resultados intermediários que se assemelham a ambos os grupos, com concentrações de 8,43±3,85 e 9,18±2,82ng/mL, respectivamente. As tentativas de ajustes no proestro foram incapazes de melhorar a qualidade folicular e minimizar a função luteal pré-ovulatória, assim como não incrementaram a morfologia do CL e a função luteal pós-ovulatória, sugerindo que em animais cíclicos mestiços protocolos de IATF com a utilização de uma única dose PGF2α e sem o suporte gonadotrófico da eCG parece promover adequada resposta folicular e luteal.(AU)

The study aimed to evaluate the effect of one or two prostaglandin doses F2α (PGF2a) with or without equine chorionic gonadotropin (eCG) in the follicular dynamics, the preovulatory luteal function, as well as the structural and functional characteristics post-ovulatory of the corpus luteum (CL) in cyclic crossbred females subjected to a fixed time artificial insemination (FTAI) protocol. For this, 29 multiparous 3/4 Gyr x Holstein cows were subjected to transrectal ultrasound examination (US) and upon detection of CL initiated a FTAI protocol on day called zero (D0) by the insertion of progesterone implant (P4) associated with the application of 2.0mg estradiol benzoate. On D7, these animals received 12.5mg of dinoprost tromethamine. At D9 happened the removal of the P4 devices and was applied 0.6mg of estradiol cypionate. At that time, the females were divided into the following treatments: control group (n=7) - which received 2.5mL of saline solution, 2PGF group (n=7) - received 12.5mg of dinoprost tromethamine, eCG group (n=7) - was administered 300IU eCG and eCG+2PGF group (n=8) - which received 300 IU eCG and 12.5mg of dinoprost tromethamine. To assess follicular dynamics were performed US scans B-mode and power doppler (Mindray Z5, Shenzhen, China) each 12h on D7 until the time of ovulation or until 96h after removal of the P4 implants, considering the follicular diameter (DFOL), the area of the follicular wall (AFOL) and the blood perfusion area of the follicular wall (VFOL). Concomitant with each test, blood samples were collected to determine the serum concentration of P4 preovulatory by chemiluminescence methodology. In D24 had held US B-mode and doppler to analyse the luteal diameter (DCL), luteal area (ACL) and blood perfusion area CL (VCL). Also, a blood sample was collected to determine the serum concentration of P4 post-ovulatory. All data was evaluated by Two-way ANOVA and repeated measures analysis considering the effects of eCG, 2PGF and eCG*2PGF, P<0.05. There was not significant difference between the synchronization protocols for DFOL, AFOL and VFOL variables over time of follicular dynamics. Experimental groups had a serum concentration of P4 preovulatory similar in every moment of evaluation. There wasn't distinction of ACL and VCL between hormone treatments. However, the eCG group showed a tendency (P=0.08) to present higher DCL compared to the 2PGF and 2PGF+eCG groups. In addition to these findings, there was also a tendency (P=0.07) to higher concentrations of P4 on D24 of the protocol in the animals of the eCG group (11.00±3.32ng/mL) compared to the 2PGF group (6,37±1.31ng/mL), meanwhile the Control and 2PGF+eCG showed intermediate results that resembled both groups, with concentrations of 8.43±3.85 and 9.18±2.82ng/mL, respectively. Attempts to adjust proestrus were unable to improve follicular quality and minimize preovulatory luteal function, nor did they increase CL morphology and post-ovulatory luteal function, suggesting that in cyclic animals, FTAI protocols using a single PGF2α dose and without the gonadotrophic support of eCG seems to promote adequate follicular and luteal responses.(AU)

Development of fixed-time artificial insemination protocols for locally adapted Curraleiro Pé-Duro cows / Desenvolvimento de protocolos de inseminação artificial em tempo fixo em vacas da raça localmente adaptada Curraleiro Pé-Duro

Two experiments were conducted aiming to evaluate the effects of two ovulatory inducers (Exp.1) and equine chorionic gonadotropin (eCG; Exp.2) on follicular and luteal dynamics in a fixed-time AI (FTAI) protocol in locally adapted Curraleiro Pé-Duro cows. In Exp. 1 multiparous cows (n=12) received an intravaginal device containing 1g of progesterone (P4) for 8 days and 2mg of estradiol benzoate (EB) intramuscularly (IM) at device insertion (Day 0). At device removal (Day 8) 0.150mg of Sodium D-Cloprostenol was administered IM and the cows were randomly assigned to receive 1mg of EB (EB8) or 1mg of estradiol cypionate (EC8) IM, or to not receive any ovulatory inducer (Control). All the animals participated in all treatments (crossover). The interval from P4 removal to ovulation was shorter and less variable in the EB8 treatment group (P≤0.05). In Exp. 2 (crossover), multiparous cows (n=12) received the same hormonal treatment as the EB8 group in Exp.1. At device removal (Day 8) cows were randomly assigned to receive 300UI of eCG IM or to not receive eCG (Control). No difference was ascertained on follicular and luteal parameters in Exp. 2 (P>0.05). We concluded that EB can be used as the ovulatory inducer (Exp. 1) in a FTAI protocol in Curraleiro Pé-Duro cows. However, eCG (Exp. 2) was not able to stimulate follicular and luteal development. This result is probably due to the adaptive capacity of Curraleiro Pé-Duro cows that maintained a satisfactory body condition score even in dry and hot environments.(AU)

Foram realizados dois experimentos com o objetivo de avaliar o efeito de dois indutores da ovulação e da gonadotrofina coriônica equina (eCG) na dinâmica folicular e luteal, em um protocolo de inseminação artificial em tempo fixo (IATF) em vacas localmente adaptadas da raça Curraleiro Pé-Duro. No experimento 1, vacas pluríparas receberam um dispositivo intravaginal contendo 1g de progesterona (P4) durante oito dias e 2mg de benzoato de estradiol (BE) intramuscular (IM) no momento da inserção do dispositivo (dia zero). Na retirada do dispositivo (dia oito), as vacas receberam 0,150mg de D-cloprostenol sódico IM e foram separadas aleatoriamente para receber 1mg de BE IM (BE8) ou 1mg de cipionato de estradiol IM (CE8), ou nenhum indutor da ovulação (controle). Todos os animais participaram de todos os tratamentos (crossover). O intervalo entre a retirada da P4 e a ovulação foi menor e menos variável no tratamento BE8 (P≤0,05). O momento da ovulação foi mais precoce e mais concentrado nos animais do grupo BE 8. No experimento 2 (crossover), vacas pluríparas receberam o mesmo tratamento hormonal do grupo BE8 do experimento1. Na retirada do dispositivo (dia 8), as vacas foram separadas aleatoriamente para receberem 300UI de eCG IM, enquanto o controle não. Não houve diferença nos parâmetros foliculares e luteais avaliados no experimento 2 (P>0,05). Em conclusão, o BE pode ser utilizado como indutor da ovulação (experimento 1) em protocolos de IATF em vacas Curraleiras Pé-Duro. Entretanto, o eCG (experimento 2) não foi capaz de estimular o desenvolvimento folicular e luteal. Esse resultado é devido provavelmente à capacidade adaptativa das vacas Curraleiras Pé-Duro em manter uma condição corporal satisfatória mesmo em condições de clima seco e quente.(AU)

Immunohistochemical characterization of telocytes in rat uterus in different reproductive states

Telocytes are a new type of interstitial tissue cells that were recently discovered in the myometrium of nonpregnant uteri and have been suggested to modulate uterine contraction. This work was conducted to extend this research on uteri at different reproductive states and verify whether the morphology and number of telocytes are modified. This study included 24 apparently healthy female albino rats equally classified into four groups: immature rats, adult nonpregnant rats, pregnant rats, and postpartum rats. The middle one-third of the right uterine horns were processed for H and E staining and immunohistochemical detection of telocytes using a c-kit antibody. The count of c-kit-positive telocytes per high-power field in both the endometrium and myometrium was determined and statistically analyzed. C-kit-positive telocytes were detected in the endometrium, mainly around the endometrial glands, and in the myometrium. They were oriented parallel to circular smooth muscles while being located predominately on the boundaries of longitudinal muscle bundles. Immature uteri contained a small number of telocytes in both the endometrium and myometrium that significantly increased in adult nonpregnant uteri. Pregnant uteri showed further significant increase in endometrial telocytes but a significant decrease in myometrial telocytes, possibly to prevent preterm delivery. Postpartum uteri showed the highest count of myometrial telocytes, which could reflect their role in postpartum involution. Telocytes are present in both the endometrium and myometrium of the rat uterus in different reproductive states. Their functions in the endometrium seem to be glandular support and stromal cell communication, whereas in the myometrium they possibly initiate and coordinate myometrial contraction

Effects of echinomycin on endothelin-2 expression and ovulation in immature rats primed with gonadotropins

Echinomycin is a small-molecule inhibitor of hypoxia-inducible factor-1 DNA-binding activity, which plays a crucial role in ovarian ovulation in mammalians. The present study was designed to test the hypothesis that hypoxia-inducible factor (HIF)-1alpha-mediated endothelin (ET)-2 expressions contributed to ovarian ovulation in response to human chorionic gonadotropin (hCG) during gonadotropin-induced superuvulation. By real-time RT-PCR analysis, ET-2 mRNA level was found to significantly decrease in the ovaries after echinomycin treatment, while HIF-1alpha mRNA and protein expression was not obviously changed. Further analysis also showed that these changes of ET-2 mRNA were consistent with HIF-1 activity in the ovaires, which is similar with HIF-1alpha and ET-2 expression in the granulosa cells with gonadotropin and echinomycin treatments. The results of HIF-1alpha and ET-2 expression in the granulosa cells transfected with cis-element oligodeoxynucleotide (dsODN) under gonadotropin treatment further indicated HIF-1alpha directly mediated the transcriptional activation of ET-2 during gonadotropin-induced superuvulation. Taken together, these results demonstrated that HIF-1alpha-mediated ET-2 transcriptional activation is one of the important mechanisms regulating gonadotropin-induced mammalian ovulatory precess in vivo.

Sincronização da ovulação utilizando FSH em substituição à eCG em cabras / Ovulation synchronization using FSH replacing eCG in goats

The effect of substitution of equine chorionic gonadotropin (eCG) by follicle stimulating hormone (FSH) in synchronization protocols of ovulation in dairy goats was evaluated. Twelve goats received intravaginal sponges impregnated with 60mg of medroxyprogesterone acetate (MAP) for 10 days. The sponges were removed and the animals were distributed into two groups (G): G1 (n=6) treated with 0.5mL of a synthetic analogue of PGF2 α and 100 IU of eCG for each 10kg weight, intramuscular injection (IM); and G2 (n=6) treated with 0.5mL of a synthetic analogue of PGF2α and 20mg of FSH (IM). All animals were monitored for estrus detection with aid of a ruffian after sponge removal. The ovarian dynamics were analyzed by ultrasound, since six hours after sponge removed. Each animal was analyzed in time elapsed of six hours until 12 hours after ovulation detection. For data analyses, the Wilcoxon test and variance analyses were used. There was not difference between the analyzed parameters (P>0.05). In this way, eCG can be replaced by FSH in synchronization protocols of ovulation in dairy goats.

Dinámica folicular en yeguas paso fino colombiano medido por ultrasonografía en la Sabana de Bogotá / Follicular dynamics in mares paso fino colombiano at Sabana of Bogotá Colombia

Este estudio surgió como medio para obtener un parámetro de crecimiento diario folicular, momento de la ovulación, y determinar cuál es el ovario con mayor número de ovulaciones en la yegua de Paso Fino Colombiano con el fin de aportar algunos datos de dinámica folicular en la yegua de esta especie, ya que la información disponible en la literatura data generalmente en otras razas y condiciones diferentes al trópico. Para el desarrollo de este proyecto se tomaron como muestra cincuenta (50) yeguas ubicadas en la sabana de Bogotá: (Chía – Cundinamarca), a una altura de 2.652 metros sobre el nivel del mar, temperatura promedio de 12° C y pluviosidad 1.500 mm; la edad de los animales osciló entre los cinco y diez años. Se realizaron ecografías periódicas día por medio por palpación rectal, con el fin de realizar un seguimiento del crecimiento folicular una vez detectado un folículo dominante (>30 mm de diámetro), hasta su ovulación. Los datos fueron analizados por medio de estadística descriptiva, con desviación estándar, además de una prueba F para varianzas desiguales e iguales para determinar diferencias en el tamaño folicular a la ovulación y crecimiento diario folicular entre el ovario derecho e izquierdo. Teniendo como resultado que la yegua de paso fino colombiano tiene un crecimiento diario folicular de 2,04+/-0,63 mm, un tamaño folicular a la ovulación de 41,34+/-2,14 mm, y que de los cincuenta ciclos estrales analizados el 60% fue por el ovario izquierdo y el 40% restante por el ovario derecho. Las diferencias en cuanto a tamaño folicular a la ovulación no fueron significativas (P>0,50) entre los ovarios, mientras que para el crecimiento diario folicular sí hubo diferencia significativa (P<0,02) lo que significa que los folículos del ovario derecho tuvieron un crecimiento mayor a los del ovario izquierdo. Con estos resultados se obtuvo que la yegua de paso fino colombiano se comporta reproductivamente de manera similar a las demás razas, dentro de las condiciones de trópico establecidas en este estudio...

[Effect of ultrasound on parthenogenic activation of mouse oocyte]

Artificial stimulation of mouse oocyte, in the absence of sperm contribution, can induce its parthenogenic activation of oocyte. Ultrasound is one of the newest methods for artificial activation of mammal oocytes, and its successful utilization in pig oocyte activation has been recently reported. Our objective was to assess the effect of ultrasound on mouse oocyte activation. Our groups included1 control group, 3 experimental groups consisting of 1, 2 and 3 repetitions of ultrasound exposure, and 3 sham groups handled similar to experimental groups but ultrasound system was off during treatments. In experimental groups, adult female NMRI mice at the interval between pregnant mare serum gonadotropin [PMSG] and human corionic gonadotropin [hCG] injections, were exposed to continuous ultrasound with 3.28 MHz frequency and peak intensity [I pk]=355 mW/cm2. Sixteen hours after injection of hCG, the mice were euthanized and their oocytes were collected; thereafter, parthenogenic oocytes were counted. Data analysis using the ANOVA test shows a significant increase in the number of parthenogenic oocytes in mice with 3 overall exposures to ovarian ultrasound [p<0.05]. A significant decrease in the number of metaphase II [MII] oocytes numbers was also seen in mice treated with ultrasound [p<0.05]. Ultrasound is thought to induce pores generation in oocyte membranes and provides an easier inward transport of Ca++ into oocytes. This phenomenon can induce meiosis resumption in immature oocytes. With increased exposure repetitions from 1 to 3 times and greater Ca++ arrival, oocytes can be parthenogenetically activated

[Mechanism of lithium's effect on follicular development of the rat ovary]

It has been shown that lithium chloride [LiCl], an effective drug for the treatment of bipolar disorder, has side effects on the female reproductive system. In this study, cellular and histological effects of lithium chloride on the development of ovarian follicles in immature female rats were investigated. To induce ovarian follicular development, twenty-three day old immature female rats were injected with 10 IU of pregnant mare serum gonadotropin [PMSG], followed by four doses of LiCl [250 mg/kg/dose] each injected every 12 hours, starting from the time of the PMSG injection. The rat ovaries were removed 48 hours after the PMSG injection and prepared for histological, immunohistochemical, and DNA laddering studies. Control immature female rats received only PMSG, while sham treated rats received PMSG and physiological serum [lithium vehicle]. Our results showed that in the ovaries of LiCl-treated rats there were neither large antral follicles [800-1000 micro m] nor fewer medium sized follicles [400-800 micro m] but a increased number of atretic follicles compared to those in the control rats. The induction of atresia in the ovaries of LiCl-treated rats was further confirmed by the presence of DNA fragmentation. Looking at the cellular levels, lithium extremely significant [p<0.0001] increased the number of TUNEL-positive cells in the granulosa layer of the antral follicles. Taken together, our results suggest that lithium may decrease folliculogenesis by inducing apoptosis in the antral follicles

[Expression of GSK-3 beta and beta-catenin proteins in the PMSG stimulated rat ovary]

The ovary is an example of a developing tissue in which developmental prosses occur throughout reproductive life. We investigate the expression of GSK-3 beta and beta-catenin-Wnt pathway molecules-in the rat ovary during follicular development. To induce follicular growth and development, 23 days old immature female rats were injected with 10 IU of PMSG. Forty and forty-eight hours after stimulation with PMSG, the sera was collected for hormone assay, and the ovary were dissected and prepared for immunohistochemistry[IHC[and western blot[WB]. Our IHC and WB analysis showed that the expression of pGSK-3 beta and active beta-catenin was increased in PMSG primed rats in compared to those of intact groups. However, the total GSK-3 beta and beta-catenin proteins remain unchanged. The results indicate this idea that gonadotropin [PMSG], stabilized beta-catenin in GSK-3 beta independent manner in the rat ovary during the follicular development

[Effects of tempol on in vitro development of mouse embryos under oxidative stress]

Etiologically, oxidative stress can be considered as one of the reasons for defective embryonic development which leads to developmental arrest due to necrosis or apoptosis. Under in vivo conditions, multiple mechanisms act to protect the embryo against reactive oxygen species [ROS], but under in vitro conditions most of these mechanisms are absent leading to higher levels of ROS in the culture medium. The objective of this study was to compare the antioxidant effects of Tempol, 4-hydroxy-2, 2, 6, 6-tetramethylpiperidine-l-oxyl, a permeable synthetic antioxidant, on mouse preimplantation embryonic development in vitro conditions in the presence or absence of oxidative stress. Mature oocytes from mouse were retrieved following ovarian stimulation by the administration of Pregnant Mare Serum Gonadotropin [PMSG] and hCG. Upon in vitro fertilization, the zygotes were cultured in different groups in HTF medium containing 4 mg/ml BSA. To study the effects of oxidative stress on embryo development, the zygotes were cultured for an hour in a medium containing different concentrations of H[2]O[2]. After washing, the zygotes were transferred to the culture plate. The zygotes were later placed in the media containing different concentrations of Tempol following their culture in 10 microM H[2]O[2] for one hour to study the effects of different concentrations of the substance in the absence of other oxidative stresses. The data were later compared and statistically analyzed. The pre-implantation embryonic development decreased significantly in the case group, compared to the control group after a short exposure to H[2]O[2], - the effect being more noticeable in higher concentrations. Tempol reduced the impairments resulting from the oxidative stress to some extent. Under in vitro conditions and a concentration of 0.5 microM, Tempol improved embryonic development quality, quantitatively and morphologically. Tempol increased the percentage of two-cell embryos from 91.78% in the control group to 96.99% [p < 0.05], blastocysts from 67.80% in the controls to 81.33% [p < 0.05] in the cases, and significantly decreased embryonic arrest from 32.19% in the controls to 18.67% in the cases [p < 0.05]. ROS has a major role in embryonic arrest, witnessed in embryo cultures in vitro conditions. The present study showed that supplementation of embryo cultures with Tempol improved the embryonic development. It seems that addition of permeable synthetic antioxidants, such as Tempol, to embryo cultures could protect embryos from oxidative damage and improve embryonic development

Efeito do eCG ou benzoato de estradiol associado ao norgestomet na taxa de concepção de vacas de corte submetidas à IATF no pós-parto / Effect of eCG or estradiol benzoate associated with norgestomet on the conception rate of beef cows submitted to FTAI in the postpartum

Este estudo objetivou comparar as taxas de concepção, em vacas de corte no período pós-parto (PPP), tratadas com Gonadotrofina Coriônica Eqüina (eCG) ou Benzoato de Estradiol (BE), após o uso de Norgestomet submetidas à inseminação artificial em tempo fixo(IATF). A hipótese é que a administração de eCG ou BE aumenta as taxas de concepção. Vacas Nelore (n=138) e Brangus (n=63), lactantes,entre 26 e 118 dias do PPP, foram divididas em três grupos homogêneos e dentro de cada grupo subdivididas em dois blocos, um com um PPP < 45 dias (PPP1; n=107) e outro > 45 dias (PPP2;n=94). Todas as fêmeas receberam um implante auricular contendo 3mg de Norgestomet (Crestar®), seguido pela administração de 5mg de Valerato de Estradiol. Os implantes auriculares foram mantidos durante 10 dias. Na retirada dos implantes, as fêmeas receberam 1mL de solução fisiológica (n=68; Grupo Controle) ou 500 UI de eCG (Folligon®; n=67; Grupo eCG) ou 1mg de Benzoato de Estradiol (Index; n=66; Grupo BE) 24 horas após a retirada do implante. AIATF foi realizada 54 horas após a retirada do implante. O diagnóstico de gestação foi realizado por ultra- sonografia 30 dias após a IATF. Houve interação entre os tratamentos e o PPP. No PPP1, a taxa de concepção foi maior no grupo eCG comparado ao BE (47,22% vs.15,38%; P<0,01). No PPP2, as taxas de concepção dos grupos eCG eBE foram maiores que no grupo controle (41,93%, 44,44% vs.22,22%; P<0,01). Conclui-se que vacas, com até 45 dias do PPP, o eCG associado ao Norgestomet aumenta as taxas de concepção.

This study aimed to compare conception rates at the post partum period (PPP) in beef cows which were administered either Equine Chorionic Gonadotrophin (eCG) or Estradiol Benzoate (EB) after Norgestomet and submitted to fixed-time artificial insemination(FTAI). The hypothesis was that the administration of eCG or E Benhances the conception rate. Lactating Nelore cows (n=138) and Brangus (n = 63), between 26 and 118 days of post partum period(PPP) were divided into three homogeneous groups, and subdividedin two blocks, inside each group one of them with a PPP d” 45 days (PPP1; n=107) and another with a PPP > 45 days (PPP2; n=94). Allcows received an auricular implant containing 3mg Norgestomet(Crestar®), followed by the administration of 5mg Estradiol Valerate.The auricular implants were kept during 10 days. Cows received 1mL saline solution (n=68, Control Group) or 500IU eCG (Folligon®;n=67; eCG Group) or 1mg Estradiol Benzoate (Index, n=66; EB Group) 24 hours after the removal of the implant. FTAI was made 54 hours after the implant removal. The pregnancy diagnosis was carried through ultrasonography 30 days after FTAI. There was an interaction between treatments and PPP. In the PPP1, the conception rate was higher in the eCG Group than in the EB Group (47.22% vs.15.38%; Pd”0.01). In the PPP2, the conception rates of the eCG and EB Groups were higher than in the Control Group (41.93%, 44.44%vs. 22.22%: Pd”0.01). It was concluded that in cows up to 45 days of PPP, the eCG associated with Norgestomet enhances the conception rates.

Effect of the dose of equine chorionic gonadotrophin [ECG] on ovarian response and embryo recovery in Egyptian baladi goats

The effects of doses of equine chorionic gonadotrophin [eCG] for a simple superovulation method in does [Goats] were investigated on ovulation rate, proportions of fertilized oocytes and normal embryos at recovery. The does were treated with intravaginal sponges impregnated with medroxy progesterone acetate [MAP] for 12 days. For superovulation, an intramuscular injection of eCG was given at 24 h before sponge removal. Does were divided into nine equal groups [n = 5] A, B, C, D, E, F, G, H and I. Each received 100, 200, 300, 400, 500, 600, 700, 800 and 900 IU of eCG respectively. All does were naturally inseminated by four healthy fertile bucks. Laparotomy and embryo recovery were done for does on day six. The mean numbers of corpora lutea of nine groups were [2.8 +/- 4.34, 4.4 +/- 0.55, 2.8 +/- 1.10, 4.2 +/- 1.79, 6.8 +/- 2.39, 9.6 +/- 4.45, 6.8 +/- 5.81, 6.6 +/- 3.05 and 5.8 +/- 5.17] respectively. While mean of unovulated follicles were counted after laparotomy for nine groups respectively [3.4 +/- 1.14, 5.8 +/- 1.30, 7.0 +/- 2.35, 5.6 +/- 1.82, 6.8 +/- 2.05, 7.2 +/- 1.92, 9.8 +/- 4.71, 11.2 +/- 5.02 and 10.6 +/- 3.58]. On the other hand the percentage of normal embryos were 0, 15.0, 42.86, 42.11, 54.84, 66.67, 48.64, 38.0 and 45.24] of normal embryos. The present study indicates that an appropriate eCG dose would be 600-700 IU for a simple superovulation method in does pre-treated with Progestagen-impregnated vaginal sponges

Nitric oxide acts through different signaling pathways in maturation of cumulus cell-enclosed mouse oocytes

Nitric oxide [NO] have a dual action in mouse oocyte meiotic maturation which depends on its concentration, but the mechanisms by which it influences oocyte maturation has not been exactly clarified. In this study different signaling mechanisms which exist for in vitro maturation of meiosis was examined in cumulus cell-enclosed oocytes [CEOs] after injection of pregnant mare's serum gonadotropin [PMSG] to immature female mice. The CEOs were cultured in spontaneous maturation and hypoxanthine [HX] arrested model. Sodium nitroprusside [SNP, an NO donor, 10mM] delayed germinal vesicle breakdown [GVBD] significantly during the first 5 hrs of incubation and inhibited the formation of first polar body [PB1] at the end of 24 hrs of incubation. SNP [10-5M] stimulated the meiotic maturation of oocytes significantly by overcoming the inhibition of HX. Sildenafil [a cGMP stimulator, 100 nM], had a significant inhibitory effects on both spontaneous meiotic maturation and HX-arrested meiotic maturation. Forskolin [an adenylate cyclase stimulator, 6 micro M] and SNP [10mM] had the same effects on GVBD. Forskolin reversed the SNP [10-5M] stimulated meiotic maturation. These results suggest that differences in pathways are present between SNP-inhibited spontaneous meiotic maturation and SNP-stimulated meiotic maturation in mouse oocytes

Efecto de la sincronización del estro con prostaglandina-f2a vs CIDR + 500 UI de eCG en ovejas bergamacia durante el inicio de la fase luteal / Effect of estrus synchronization with prostaglandins-F2a vs CIRD + 500 IU of eCG in bergamacia ewes during early luteal phase

Con el objeto de evaluar los efectos de la sincronización del estro con PGF2a vs CIDR + 500 UI de eCG, sobre el tiempo de presentación de estro, ovulación y las concentraciones plasmáticas de hormonas esteroidales durante el inicio de la fase luteal en ovejas, se seleccionaron 14 hembras Bergamacia distribuidas en dos grupos: el Grupo uno (Control), sometido a dos aplicaciones de prostaglandina-F2a (PG), con un intervalo de nueve días, y el Grupo dos, tratado con el dispositivo intravaginal impregnado con progesterona (CIDR) durante 12 días y 500 UI de eCG. La presentación del estro fue de 100%. Entretanto, el intervalo estro-ovulación fue de 36,0 ± 0,72 horas en el grupo dos. El grupo control tratado con PG presentó un intervalo estro-ovulación de 53,42 ± 3,0 horas (P<0,001). Hubo diferencia significativa (P<0,01) en las concentraciones plasmáticas de P4 entre los tratamientos. Los animales del Grupo dos presentaron aumentos significativos (P < 0,01) en las concentraciones plasmáticas de P4, desde el sexto hasta el décimo día después de la ovulación, comparados con las concentraciones de los animales control. Del mismo modo, también se constató una diferencia significativa en la interacción tratamiento y día (P< 0,05). Las concentraciones de E2 en el plasma sanguíneo fueron estadísticamente diferentes (P< 0,001) entre el grupo control y el grupo sincronizado con el CIDR + eCG. Fueron observadas diferencias significativas (P< 0,001) en las concentraciones plasmáticas de E2 entre los tratamientos después del día ovulatorio (día cero), siendo además constatada diferencia significativa entre los días (P< 0,001) y en la interacción tratamiento y día (P< 0,05). Se puede concluir que, la sincronización del estro en hembras Bergamacia, utilizando el CIDR + eCG disminuyó el intervalo estro-ovulación y provocó aumento de las hormonas esteroidales en plasma.

An experiment was conducted to investigate the effects of estrous synchronization with PGF2a vs cidr + 500 IU of eCG on interval to estrus, ovulation and steroidal hormones plasma concentrations during early luteal phase in sheep. Fourteen ewes were treated, distributed in two groups: Group 1 (Control), synchronized with two injections of prostaglandin-F2a (PG), given 9 days apart, and Group 2, was treated with CIDR for 12 days and 500 IU of eCG. Estrous presentation was 100%. The interval estrus-ovulation was 36.0 ± 0.72 h in Group two. The control group presented an interval estrus-ovulation of 53.42 ± 3.0 hours (P<0.001).There were significant difference (P < 0.01) in P4 plasma concentrations among treatments. In Group 2, the animals showed significatives increases (P < 0.01) in P4 plasma concentrations during day 6 until day 10 when compared with control animals. It was observed significative difference at interaction treatment by day (P < 0.05). E2 plasma concentrations were statistically differents (P < 0.001) among control group and synchronized group with CIDR + eCG. There were significant difference (P < 0.001) in E2 plasma concentrations among treatments after ovulation day (Day 0) with significative difference among days (P < 0.001) and interaction treatment x day (P < 0.05). These results indicated that estrous synchronization in Bergamacia females, using CIDR + eCG diminished interval estrus-ovulation and elicit higher levels of steroidal hormones in plasma.

Evaluación del tratamiento superovulatorio con extracto de hipófisis equina en yeguas criollas / Evaluation of superovulatory treatment with extract of equine hypophysis in creole mares

Se evaluó la respuesta superovulatoria en yeguas criollas a las cuales se aplicó extracto de hipófifis equina (EHE)en dos diferentes dosis, comparada con FSH de origen porcino (Folltropin V). Se tomaron 20 yeguas criollas Colombianas cíclicas con edades entre 2 y 8 años (5,3 años en promedio) las cuales fueron seleccionadas y asignadas al azar en 4 grupos así: Grupo 1 También llamado grupo control (T1): se les aplicó 5cc suero fisiológico IM (a.m., p.m.); grupo 2 (T2): 6,25mg Foltropin-v (FSH-P) IM (a.m., p.m.); Grupo 3 (T3): 8,3mg EHE IM (a.m., p.m.); Grupo 4 (T4): 12,5mg EHE IM (a.m., p.m.). Los tratamientos se iniciaron 7 días después de haber dectado una ovulación mediante ultrasonografía, el día 8 se aplicaron 12,5 mg PgF 2alfa vía IM. El tratamiento con EHE se suspendió cuando la mayoría de los folículos alcanzaron un tamaño > 35mm, momento en el cual se aplicaron 2500 UI de hCG vía IV, seguida de inseminación artificial. Entre el día 7 u 8 se realizaron las colectas de embriones mediante lavado uterino de circuito cerrado. Los datos se analizaron mediante un análisis de varianza por una vía (ANOVA), determinando el promedio de crecimiento folicular entre el día del inicio del tratamiento y la ovulación, el número de fóliculos preovulatorios con un tamaño > 35mm, el número de días de tratamiento y la cantidad de embriones colectados en cada grupo. Se realizó una prueba de rango o comparción múltiple de Duncan para observar las diferencias entre grupos y se determinó por análisis de relación el estado y la calidad de los embriones en cada tratamiento para determinar la viabilidad. Los resultados obtenidos demostraron que en T4 se desarrolló un mayor número de folículos > a 35mm en comparación con los demás grupos (P<0.005) en 7,4 días de tratamiento, mayor rata de crecimiento folicular (3.01 mm/día), mayor número de ovulaciones por tratamiento (2.8) y mayor número de embriones colectados por yegua (1,6), siendo este (T4) el tratamiento de mejores resultados res.

Effect of recombinant somatotropin with or without PMSG on postpartum multiparous buffalo in Egypt

This study was carried out on ten Egyptian female multi parous buffaloes suffering from prolonged postpartum period with no estrous signs and low serum progesterone level. Animals were randomly divided into two groups. Females in both groups were injected subcutaneously with 500 mg recombinant bovine somatotropin "rbST",one group received a single intramuscular dose of PMSG [2000 i.u.] 7 days later. Blood samples were collected one week before treatment and for four consecutive weeks after treatment. The levels of progesterone, insulin-like growth factor- 1[IGF-1] and total cholesterol were analysed. Injection of rbST with or without PMSG elevated serum level of IGF-1 for the first two weeks post-injection then declined during the 3rd and 4th weeks compared to pretreatment levels. Both serum progesterone and total cholesterol levels increased throughout the 4 weeks post-injection as compared with their corresponding control values before treatments. In addition, rbST increased milk yield significantly as well as motivated the females to resume cyclic activity of the ovaries [4 / 5 in animals treated with rbST plus PMSG and 3/5 in those treated with rbST alone]. The conception percent improved

Effect of ovine follicular fluid peptide on ovarian responses and other organ weights in rats, Rattus norvegicus Berkenhout 1769.

The present study was aimed to study the effect of an ovine follicular fluid peptide on ovarian follicle and good oocyte numbers and weights of ovary, uterus, liver, pancreas and kidney in rats, R. norvegicus. A 30.1 kDa peptide was isolated from ovine follicular fluid by ammonium sulphate precipitation and then gel filtration. The peptide was tested at various levels in normal (22 and 36 day-old), superovulated (29 day-old) immature and 121-day old mature rats on the ovarian responses and other organ weights. The isolated peptide inhibited the growth of antral follicles in normal and superovulated rats. Ovarian, uterine weight and recovery of good oocytes were reduced when the peptide was administered at 100 microg dose. The peptide had no effect on kidney, liver, pancreas weight and recovery of preantral follicles.

[Evaluation of acid phosphatase activity alterations in mouse ovary after ovarian hyperstimulation in early stages of pseudo and normal pregnancies until implantation time]

Acid phosphatase [ACP] is a lysosomal enzyme which contributes in ovarian metabolic functions such as oocyte maturation, resumption of mitotic divisions, germinal vesicle breakdown and ovulation. It digestes the corpus lutetium and helps the atresia of follicles by autophagia and hetrophagia activities. Considering the hormonal control of this enzyme, the present study was designed to deterimine the ovarian ACP activity after ovulation stimulation by the administration fo PMSG and hCG during preimplantation period. For this purpose a number of 6 to 10-week old female NMRI mice were selected and randomly divided into control and hyperstimulated groups after the administeration of PMSG and hCG, and later to pregnant and pseudopregnant groups. The mice were rendered pseudopregnant by mechanical vaginal stimulatin. Five mice in each group were sacrificed by cervical dislocation at the first to the sixth day of pregnancy for biochemical assays. The ovarian samples were obtained and were hemogenated and centrifuged at 14000 g. The activity of the enzyme was determined using paranitrophenyl phosphate as substrate and later the specific activity of the enzyme was calculalated according to the amount of total protein. The data were analysed by Mann Whitheny test. Statistical significance was indicated by a P value less than 0.05. For hisotchemical evaluations, the sampels were obtained from one of the ovaries in each mouse and then 5 micro m thick cryosections were prepared. Cryosections were stained by Goumory method. The ACP activity of ovarian tissues in the first day of pregnancy in the normal pregnant and pseudopregnant control groups, hyperstimulated normal pregnant and pseudopregnant groups were 0.34 +/- 0.04 IU/mg, 0.39 +/- 0.04 IU/mg, 0.4 +/- 0.08 IU/mg, 0.45 +/- 0.01 IU/mg respectively and in the fourth day were 0.69 +/- 0.1 IU/mg, 0.61 +/- 0.06 IU/mg, 1.09 +/- 0.10 IU/mg and 0.79 +/- 0.05 IU/mg. The results showed that biochemical findings correlated with histochemical observations. The ACP reaction changes were seen mainly in granulosa cells with a minimum enzyme activity in the first day [zero activity] and a maximum activity in the fourth day of pregnancy [+3]. The increased ACP activity on the 3[rd] 4th days of pregnancy, may be due to the esteroidogenic activity of granulosa cells. Also, the results showed that ovarian hyperstimulation could not change the pattern of ovarian ACP activity during early stages of pregnancy. More research is required in this area for a better understanding of the processes