Fixed-time artificial insemination protocols on brazilian locally adapted breed gilts on ovulatory response and embryo production.

The aim of this study was to use estrus synchronization protocols to favor fixed-time artificial insemination and consequently fixed-time embryo collection, and increase embryo production using eCG, in gits. In a cross over design, nine Piau breed gilts were subjected to 18 days of oral progesterone; P4 group did not receive any further; GnRH group received 25µg of GnRH 104 hours after the final application of P4; and eCG+GnRH group received 1000IU of eCG 24 hours after the final P4 in addition to GnRH for subsequent embryo collection, that was performed six days after first AI, by laparotomy. Artificial insemination was performed after 12 and 24 hours of estrus in P4 group, and 128 and 144 hours in GnRH and eCG+GnRH groups. The number of CL (8.6±3.9; 8.3±2.1; 26.7±15.0) and anovulatory follicles (4.3±3.7; 3.9±3.9; 17.2±9.5) was higher in the eCG+GnRH gilts (P<0.05). However, the use of 1000 IU of eCG reduced (P<0.05) the number of total structures (5.2±3.6; 5.1±3.1; 1.7±2.7), viable embryos (5.0±3.5; 4.8±3.3; 0.4±0.7), freezable embryos (3.6±3.4; 3.3±3.8; 0.1±0.3) and recovery rate (63.7±38.9; 58.6±24.7; 5.38±9.5). P4 and GnRH protocols were effective in the production and recovery of embryos. However, the use of 1000 IU of eCG, 24 hours after P4, was not effective in promoting the production of embryos, although the animals had superovulated.

A Design of a Time Synchronization Protocol Based on Dynamic Route and Forwarding Certification.

Time synchronization is a key technique in large-scale wireless sensor network applications. In order to tackle the problems of multi-hop synchronization error accumulation, clock frequency skew swinging, and network topology changes, a time synchronization protocol based on dynamic routing and forwarding certification (DRFC-TSP) is proposed in this paper. During the time synchronization process, a reference node with fewer synchronization hops and a more stable clock frequency is selected for every single hop, in order to obtain the best synchronization route. In this way, synchronization error accumulation can be restrained and the impact of clock frequency skew swinging on the time synchronization precision can be reduced. Furthermore, changes of the network topology can be well adapted by dynamic routing, in which the reference node is updated in every synchronization round. In the forwarding certification process, the status of nodes forwarding synchronous information outwards is authored by information exchange between neighboring nodes. Only synchronous information of the certificated nodes with a better performance can be forwarded. The network traffic can be decreased and the time synchronization precision can also be ensured, even with less energy consumption. Feasibility testing in large-scale wireless sensor networks is verified on NS2 simulation and more performances are evaluated on an embedded Linux platform.

Factors associated with pregnancy rate in fixed-time embryo transfer in cattle under humid-tropical conditions of México.

The objective was to determine the effect of some factors on pregnancy rate of fixed-time embryo transfer (FTET), in cows and heifers kept under Mexican tropical conditions. Recipients females (n=405) grazing in pastures were selected according to breed group (Zebu and crosses), parity (nulliparous and multiparous), body condition score (BCS) and the presence of a corpus luteum (CL). The females were synchronized on day 0 using a progesterone vaginal device and 2 mg estradiol benzoate (EB), two groups were established. Group 1 (conventional protocol) were animals in which the progesterone device was removed on day 7. At this time, also received an injection of 50 mg cloprostenol sodium and 1 mg estradiol cypionate. Animals also received 300 IU (heifers) or 360 IU (cows) of eCG. Group 2 (J-Synch protocol) were animals in which the progesterone device was removed on day 6. Cloprotenol and eCG injections were applied as in Group 1. Additionally, on day 9, animals of group 2 received 0.01 mg buserelin acetate. Embryo transfer of in vivo or in vitro was done on day 16 and pregnancy diagnosis was realized by ultrasonography on days 23 and 53 after FTET. Statistical analyses were carried out using Chi-square tests and logistic regression. Pregnancy rate varied between farms (P<0.05). The highest pregnancy rate was for multiparous cows (66%). The recipient utilization rate was better in the J-Synch protocol (85%), and in vivo embryos (75%) had higher pregnancy rate. The diameter of the follicle and the CL had no effect on pregnancy rate (P>0.05). However, the logistic regression determined that the only significant factor on pregnancy rate was the type of embryo. In conclusion, pregnancy rate in FTET females was higher for in vivo embryos than for in vitro embryos in cows evaluated under humid tropical conditions in Mexico.

Synchronization of germinal vesicle maturity improves efficacy of in vitro embryo production in Holstein cows.

Germinal vesicle oocytes obtained by ovum pick-up (OPU) on a random day are heterogeneous in terms of chromatin maturity, and those with an intermediate degree of chromatin compaction present higher developmental competence. We previously developed a synchronization protocol combining follicle aspiration and FSH treatment capable of increasing the percentage of oocytes with intermediate chromatin compaction (classified as GV2 oocytes; within progressive stages of chromatin compaction ranging from GV0 to GV3) at the time of OPU. In this study, we tested the capacity of a similar protocol to synchronize oocyte chromatin maturity before OPU, as well as to improve the efficacy of in vitro embryo production (IVP) in Holstein cows. In the first experiment, eight non-lactating Holstein cows were subjected to the D5/4FSH, during which all follicles larger than 2 mm were aspirated and a progesterone intravaginal device was inserted on a random day (day 0). Subsequently, four IM injections of FSH (Folltropin; 40/40/20/20 mg) were administered 12h apart on days 2 and 3, and removal of the progesterone device and OPU were performed on day 5. Of the oocytes recovered by OPU, 83.2% were at the GV2 stage. In a second experiment, eighteen non-lactating Holstein cows (Synchro group) were subjected to the D5/4FSH protocol followed by IVM/IVF, and embryo production was compared with that of other seventeen cows submitted to OPU on a random day followed by IVM/IVF (Control group). Blastocyst rate in relation to total oocytes recovered was higher in the Synchro group (37.9%) compared to the Control group (21%; P < 0.05). The percentage of good quality blastocysts morphologically selected for freezing and later transfer in relation to the total number of oocytes recovered tended to be higher in the Synchro group (27.68%) compared to the Control group (14.34%; P = 0.1). These data suggest that synchronization protocols increasing the percentage of GV2 oocytes in the population subjected to IVM/IVF can improve the efficacy of embryo in vitro production in cattle.

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)

Racionalidad de los esquemas de superovulación y sincronización en la transferencia de embriones en bovinos: ¿terapéutica basada en la evidencia o ausencia de ética / Racionalidade dos esquemas de superovulação e sincronização na transferência de embriões em bovinos: terapêutica baseada em provas ou falta de ética / Rational of superovulation and synchronization protocols for bovine embryo transfer ¿Evidence-based therapeutics or lack of ethics

Desde el inicio del uso intensivo de la transferencia de embriones (TE) en Colombia, los esquemas hormonales para la inducción de la superovulación (SOV) en las donadoras y la sincronización de las receptoras (SR), se han aplicado de conformidad con los estudios realizados en otras partes del mundo. Una gran cantidad de variaciones en los esquemas está disponible en la literatura, cuya aplicación no ha obedecido al diseño de estudios clínicos completos, sino que se basan en el principio de la utilización de productos hormonales por su presumida farmacología, sin hacer seguimiento ni de su farmacocinética ni de los potenciales efectos colaterales adversos en la fertilidad. Los esquemas utilizados podrían tener efectos colaterales asociados de manera directa o indirecta con el uso incontrolado de hormonas exógenas, o con el tiempo durante el cual las donadoras o receptoras son sometidas a los tratamientos, olvidando aspectos fundamentales de la ética en la experimentación animal. La proliferación de esquemas de SOV y de SR busca mejorar las tasas de respuesta en la producción de embriones de calidad I y II (transferibles o congelables), y aumentar las tasas de gestación de los embriones transferidos, pero los resultados no han mejorado significativamente a lo largo de los últimos 20, situación común en todos los países del mundo. En el presente trabajo se hace una revisión sobre el uso de hormonas en los esquemas de SOV y sincronización de receptoras, con énfasis en la racionalidad del uso de las hormonas y los resultados obtenidos. Finalmente, se proponen varios elementos de juicio para plantear una discusión sobre la racionalidad y la bioética de los esquemas utilizados en la TE.

Since the beginning of the use of bovine embryo transfer (ET) in Colombia, hormonal protocols for superovulation (SOV) of donors and for synchronization of recipients (RS) have been used in accordance with protocols reported elsewhere. Several variations of ET protocols have been reported most of they not based in controlled clinical studies but in the supposed pharmacological mechanisms of action of the used hormones, neither with pharmacokinetic studies nor considering the potential collateral effects on cow’s fertility. Such collateral effects could be directly or indirectly related with the uncontrolled use of exogenous hormones, or with the time-related additional open days of donor and recipients, without considering the bioethics for animal experimentation. The huge proliferation of protocols for SOV and RS aims for improving both grade I and II (transferable or freezable embryos) embryo yield per transfer, and pregnancy rates for transferred embryos, without having achieved significant improvements during the last 20 years all around the world. In the present review we critic the use of exogenous hormones in bovine ET with emphasis in the rationality of indiscriminating use of exogenous hormones and embryo yield. Finally, we suggest several arguments on the bioethics of the protocols for SOV and RS in bovine ET.

Desde o início da transferência de embriãos (TE) na Colômbia, nos esquemas hormonais para a indução da superovulação (SOV) no dadora e sincronização do receptor (SR), foram executadas de acordo com estudos realizados noutras partes do mundo. Um lote de variações nos esquemas está disponível na literatura, cuja execução não tenha obedecido a concepção dos estudos clínicos completos, sem controlo ou a sua farmacocinética ou potenciais efeitos colaterais adversos sobre a fertilidade. Os esquemas utilizados poderiam ter efeitos secundários associados direta ou indiretamente com o uso descontrolado de hormônios exógenos, ou o tempo durante o qual o doador ou receptor são submetidos a tratamento, ignorando os aspectos-chave da ética na experimentação animal. A proliferação de esquemas SOV e SR pretendem melhorar as taxas de resposta na produção de embriões qualidade I e II (transferíveis ou congeladas) eu aumento das taxas de gravidez de embriões transferidos, mas os resultados não têm melhorado significativamente ao longo dos últimos 20, uma situação comum em todos os países do mundo. Neste documento é uma revisão sobre o uso de hormônios nas formas de SOV e sincronização de receptores, com ênfase na utilização racional das hormonais e os resultados obtidos. Por último, propomos vários elementos de juízo para levantar uma discussão sobre a racionalidade e bioética dos esquemas utilizados na TE.