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The study was aimed to test the effect of the separation distance between males and females during the preconditioning period on the reproductive performance of Santa Inês ewes after the male effect. Santa Inês ewes were kept at distances of 3000 m (T1), 3 m (T2), and 300 m (T3) from rams for 60 days before starting 45-day mating seasons during the dry period (DP) and rainy periods (RP). Mating events were observed daily at 6:00 h and 16:00 h by trained personnel for one hour intervals. Estrous were scored as synchronized when observed until day 5 after breeding season start. Pregnancy diagnosis was performed by ultrasonography. In the DP, the first estrous averaged at 15.45±10.36 (T1), 9.25±6.41 (T2) and 13.05±10.24 (T3) days and in RP was 8.73±5.84 (T1), 9.30±5.62 (T2) and 6.10±5.66 (T3) days. All females cycled during both DP and RP. Estrous synchronization occurred in 20% of the females during DP (T1: 30%, T2: 15%, and T3: 15%). In the RP, estrous synchronization occurred in 40% of all females (T1: 30%, T2: 35%, and T3: 45%). The pregnancy rates in DP and RP were T1: 85%, T2: 80%, and T3: 75%. The results show that the male effect can be obtained simply by avoiding physical contact between males and females throughout the year under tropical conditions.

Avaliou-se a ação de doses reduzidas e convencionais de substâncias luteolíticas sobre parâmetros clínicos e reprodutivos de éguas. As femeas receberam intramuscularmente, 125 μg (n = 20) e 250 μg (n = 20) de Cloprostenol e 2.5 mg (n = 20) e 5.0 mg (n = 20) de Dinoprost. A temperatura retal e as frequências cardíaca e respiratória foram aferidas antes e após a administração desses luteolíticos, considerando-se ainda a ocorrência de sudorese, diarreia, cólica e prostração. Monitorou-se o estro e o desenvolvimento folicular até a ovulação, quando realizou-se a inseminção artificial. A gestação foi diagnosticada com 300 e confirmada no 60o dia. Apenas as éguas tratadas com 2,5 e 5,0 mg de Dinoprost apresentaram alteração (P < 0.05) da frequência respiratória e os demais parâmetros não foram alterados (P > 0.05). A sudorese ocorreu em 5% e 10% das éguas tratadas, respectivamente, com 2.5 mg e 5.0 mg de Dinoprost e a diarréia em apenas 5% daquelas que receberam 5.0 mg desse luteolítico. As porcentagens de estro e prenhez das éguas tratadas com 125 μg de Cloprostenol (45%/35%) e 2.5 mg de Dinoprost (50%/30%) foram menores (P < 0.05) do que os daquelas que receberam 250 μg de Cloprostenol (85%/70%) e 5 mg de Dinoprost (90%/75%). O estro e a prenhez das éguas Controle foram menores (P < 0.05) do que nas tratadas. Conclui-se que apesar de não promoverem alterações significativas dos parâmetros clínicos, as doses reduzidas não apresentam as mesmas eficiências dos tratamentos com doses convencionais para induzir o estro.

SummaryThe experiment aimed to compare conventional freezing and different vitrification protocols for cryopreservation of caprine embryos at morphological, ultrastructural, and functional levels. Caprine embryos produced in vivo were allocated randomly to three groups: (1) conventional freezing with ethylene glycol (EG); (2) dimethyl sulfoxide + EG (DMSO/EG) vitrification; and (3) dimethylformamide + EG (DMF/EG) vitrification. All groups were scored for cell viability (propidium iodide staining and ultrastructural levels) and re-expansion rate after thawing or warming. Embryos subjected to DMSO/EG vitrification showed higher cell viability (73.33%), compared with DMF/EG vitrification and conventional freezing group embryos (40.00 and 66.66%, respectively). The ultrastructural study revealed that vitrified embryos had greater preservation of cellular structure than embryos from conventional freezing with EG. DMSO/EG vitrification resulted in higher rates of re-expansion in vitro (47.36%) than DMF/EG vitrification (31.58%), and conventional freezing (25.00%). In conclusion, caprine embryos produced in vivo are better cryopreserved after vitrification than conventional freezing, therefore we conclude that DMSO/EG vitrification is the most effective protocol for cryopreservation.

The study was aimed to evaluate the effect of breeding season (BS) duration on goat reproduction during dry (DS) and rainy (RS) periods. Females were kept 300 m apart from bucks for 60 days. Females were randomly distributed among BS groups of 25 (BS25), 35 (BS35), and 45 (BS45) days during DS and RS. All females cycled, except during BS45 in DS (95%). The duration of BS did not affect estrous incidence during DS and RS. The percentage of females with two estrous was similar in RS (BS25: 30%, BS35: 35%, BS45: 35%) and DS (BS25: 25%, BS35: 30%, BS45: 25%). Three estrous were observed in BS35 and BS45 during RS (5%). First estrous (FE) and second estrous (SE) were detected from days in BS25 (FE: 1-21, SE: 7-21), BS35 (FE: 1-23, SE: 6-27), and BS45 (FE:1-23, SE: 9-20) during RS. During DS, estrous were detected within days BS25 (FE: 1-17, SE: 6-23), BS35 (FE: 1-20, SE: 6-24), and BS45 (FE: 2-21, SE: 6-21). Pregnancy rates were similar in RS (BS25: 90%, BS35: 95%, BS45: 95%) and DS (BS25: 75%, BS35: 80%, BS45: 75%). In conclusion, goat BS can be shorten despite weather without any effect on reproduction.

Neste trabalho foi avaliado o efeito do estresse calórico durante a maturação de oócitos sobre a produção in vitro de embriões ovinos. Os ovários foram obtidos em abatedouro e os oócitos colhidos de folículos de 2 a 6 mm de diâmetro. Após seleção, os oócitos, em 10 replicações, foram colocados para maturação in vitro (MIV) durante 24 horas. Os oócitos submetidos ao estresse térmico de 41º C durante 3, 6, 12, 18 e 24 horas foram posteriormente transferidos para completar a MIV a 39 ºC, mesma temperatura utilizada para maturação dos oócitos do grupo controle. O desenvolvimento dos embriões foi determinado nos dias 3, 4, 5 e 8 pós-fecundação. A avaliação da qualidade dos embriões foi efetuada através da contagem total de células coradas pelo DAPI e da determinação do número de blastômeros positivos para apoptose através do teste de TUNEL. Observou-se que o estresse térmico diminuiu (P < 0,05) a capacidade de maturação dos oócitos de acordo com o tempo de exposição à temperatura de 41º C. No grupo de oócitos incubados a 39° C, 70,70% maturou, enquanto que nos grupos expostos ao estresse térmico, apenas 45,28%, 35,17%, 12,30%, 9,74% e 4,60% maturaram, respectivamente, após 3, 6, 12, 18 e 24 horas de incubação. A duração de exposição dos oócitos ao estresse calórico foi inversamente proporcional (P < 0,05) à capacidade de desenvolvimento embrionário e diretamente proporcional (P < 0,05) ao número de blastocistos positivos para apoptose. Todavia, o efeito deletério do estresse térmico sobre a clivagem e os embriões nos estádios de 8 a 16 células e de mórula foi crescente (P > 0,05) somente até 18 horas de incubação. Os resultados permitem concluir que o estresse calórico durante a maturação in vitro de oócitos reduz a quantidade e a qualidade dos embriões ovinos produzidos in vitro determinadas pela alta incidência de apoptose. The aim of this work was to evaluate the effect of heat stress during oocyte maturation on ovine in vitro embryo production. Ovaries were collected at abattoirs and oocytes retrieved from follicles ranging from 2 and 6 mm in diameter. After selection, all oocytes, in 10 replicates, were placed in in vitro maturation (IVM) during 24 hours. The oocytes were submitted to heat stress at 41º C during 3, 6, 12, 18 and 24 hours and were further transferred to 39º C in order to complete IVM, which was the temperature of maturation of control oocytes. Embryonic development was determined on days 3, 4, 5, and 8 post-fertilization. Embryo evaluation was performed as total cell count by DAPI staining and determination of positive blastomere for apoptosis by the TUNEL assay. We observed that heat stress diminishes (P < 0.05) oocyte maturation capacity in accordance with exposure time at 41º C. In the group of oocytes incubated at 39 ºC, 70.70% matured, while in the groups exposed to heat stress at 41º C, only 45.28%, 35.17%, 12.30%, 9.74% and 4.60% matured after 3, 6, 12, 18 and 24 hours of incubation, respectively. The duration of exposure to heat stress was inversely proportional (P < 0.05) to embryonic developmental capacity and directly proportional (P < 0.05) to the number of blastocysts positive to apoptosis. However, the cleavage rate and embryonic development from 8 to 16 cells and morulae stages were affected by heat stress (P > 0.05) only up to 18 hours of incubation. The results allow the conclusion that heat stress during oocyte in vitro maturation reduces the quantity and quality of ovine embryos produced in vitro determined by the high incidence of apoptosis.

Objetivou-se com este estudo determinar a influência das estações seca e chuvosa na maturação de oócitos e produção in vitro (PIV) de embriões na espécie caprina. Os ovários das cabras nas estações seca (outubro a março) e chuvosa (abril a setembro) foram colhidos em abatedouro e transportados ao Laboratório de Biotécnicas da Reprodução da UFRPE. Os complexos cumulus oophorus (CCOs) foram colhidos pela técnica de "slicing" dos folículos entre 2 a 6 mm de diâmetro e selecionados com base na classificação morfológica. Foram realizadas 12 repetições, nas quais os CCOs foram submetidos à maturação, fertilização e cultivo in vitro dos embriões. A média e desvio padrão da taxa de clivagem foi determinada no dia 3 (D-3) e dos embriões que se desenvolveram aos estádios de 8-16 células, mórula e blastocisto foi determinada nos dias 4 (D-4), 5 (D-5) e 8 (D-8) após a fecundação, respectivamente. A quantidade de blastômeros foi determinada com o corante DAPI e os blastômeros positivos para apoptose por meio do teste de TUNEL. A produção de embriões no D-3 e mórulas foram inferiores ao obtido no período chuvoso (P < 0,05). Não apresentaram diferenças (P > 0,05) quanto às fases de maturação, fertilização, cultivo no D-4 e blastocisto. Os embriões produzidos na estação seca apresentaram maior incidência de apoptose (P < 0,05). Nas condições descritas neste estudo, os resultados permitem concluir que as fases iniciais do desenvolvimento embrionário sofrem maior impacto negativo durante a estação seca em protocolos de PIV na espécie caprina. This study aimed to determine the influence of dry and rainy seasons on oocyte maturation and in vitro production (IVP) of embryos in goats. The ovaries of does in dry (October to March) and rainy season (April-September) were collected at a slaughterhouse and transported to the Laboratory of Biotechnical Reproduction of UFRPE. The cumulus oophorus complexes (COCs) were collected by the technique of slicing of the follicles from 2 to 6 mm in diameter, and selected based on morphologic classification. We used 12 replicates, in which the COCs were submitted to maturation, fertilization and in vitro culture. The cleavage rate was determined on day 3 (D-3) and embryos that developed to 8-16 cells (D-4), morulae (D-5) and blastocyst (D-8) stages after fertilization. The number of blastomeres was assessed with DAPI staining, and the determination of apoptotic blastomeres was performed by TUNEL assay. In the dry season, D-3 embryos and morulae development was lower than that obtained in the rainy season (P0.05) were observed on oocyte maturation, fertilization, D-4 embryo yield and blastocyst development. Embryos produced during the dry season had a higher incidence of apoptosis on D- 3 and at the morulae stage (P

Background: The male effect is an attractive strategy to increase herd production by concentrating mating events and deliveries and further allowing the adoption of genetic improvement programs. It holds similar efficiency to those chemically based estrous synchronization methods, but has the advantage of being a natural method. The work was aimed to evaluate the influence of male effect on estrous induction and synchronization, pregnancy and prolificacy of nulliparous Santa Inês and Morada Nova ewes raised in Semiarid and Zona da Mata regions of Pernambuco state.Materials, Methods & Results: Santa Inês (n = 80) and Morada Nova (n = 80) females, with age from 11 to 12 months, after being evaluated and selected, were identified with plastic ear tags, weighted and maintained isolated from males, during 30 days before experiment onset, without any physical, visual, olfactive and auditive contact. Estrous events were observed twice a day (6:00 and 16:00 h) by trained personnel, during a breeding season of 60 days, and estrous were considered synchronized when detected, within first five days of breeding season. Rams of Santa Inês (n = 2) and Morada Nova (n = 2) breeds were selected based upon reproductive capacity by an andrology exam, and were marked on the externum bone region with a wax and ink (4:1) mixture, and were marked in female lots in order to identify females in estrous. After ten days of breeding season onset, rams were again marked with the same wax and ink mixture, but with a different ink color. Pregnancy diagnosis was performed on day 30 by ultrasonography and confirmed on day 60 after the last mating. The statistical analysis was performed using SAS, version 8. Differences of 5% (P < 0.05) or lower were considered significant. The results show that estrous synchronization in the semiarid region in both breeds was detected in 10% of females. The total pregnancy on first service was 45.00% and on second was 52.94%, with 85.18% of singleton and 14.81% twin deliveries, with prolificacy de 1.15 ± 0.38. In the Zona da Mata region 10% Santa Inês and 15% Morada Nova females had synchronized estrous (P > 0.05). The total pregnancy was 42.50% on first, 64.70% on second service and total delivery was 86.20% singletons, 12.06% twins and 3.33% triples with prolificacy of 1.15 ± 0.31. On both Semiarid and Zona da Mata regions, the majority of estrous events occurred between the11th and 15th day of the breeding season for Santa Inês ewes and between 6th and 10th day for Morada Nova ewes.Discussion: The occurrence of estrous, for both breeds, in both regions, were detected throughout the breeding seasons, despite most estrous detections were within the initial fifteen days, which normally happens with cycling pluriparous females, in disagreement with findings in the literature that young females display lower reproductive performance on the first breeding season. However, the sexual inexperience of young females is not equivalent to lack of male receptivity, since then, could not be responsible for late estrous onset in a breeding season of young females. In agreement with this statement, and based on the data described here, it has been described that young ewes display estrous within the initial 18 days of breeding season onset. The estrous dispersion in biostimulation programs is normally due to female cyclicity, a physiological condition that lowers the sensibility to estradiol negative feedback response, but are still responsible to the presence of males.

The present work aimed to evaluate the transcription and replication inhibitor, actinomycin D, for oocyte chemical enucleation. Cattle oocytes matured in vitro were treated with actinomycin D according to the following treatments: T1, control; T2=1.0 microg/ml for 16 h; T3=1.0 microg/ml for 14 h; T4=2.5 microg/ml for 14 h; T5=5.0 microg/ml for 14 h. The oocytes were denuded and activated during 24-26 h of maturation. Oocytes were fixed to determine the maturation status and for chromosome morphology evaluation. Furthermore, oocytes treated with actinomycin D were used for somatic cell nuclear transfer (SCNT). Parthenogenetic and SCNT embryos were fixed to evaluate the percentage of apoptotic nuclei by the TUNEL assay. The maturation (T1=90.4%; T2=82.3%; T3=79.1%; T4=83.4%; T5=74.7%), cleavage (T1=68.9%; T2=46.0%; T3=49.7%; T4=33.4%; T5=29.3%) and blastocyst rate at D8 (T1=41.1%; T2=1.8%; T3=1.3%; T4=0.9%; T5=0.0%) after actinomycin D treatment were significantly different. There was a significant chromosome uncoiling when treated with greater concentrations (2.5 and 5.0 microg/ml). After SCNT, the cleavage rate (61.3%) was similar to the actinomycin D-treated control group (61.3%) and less than the non-treated control (70.2%), although the blastocyst rate was greater in the SCNT group (11.8%) comparing with the treated control (3.6%) and less than the untreated control (38.0%). Treated parthenogenetic embryos had more apoptotic cells than the parthenogenetic controls (24.2% compared with 4.8%). However, the SCNT group using treated cytoplasts was similar from the SCNT control (9.3 compared with 13.0%). Actinomycin D treatment was efficient in blocking embryonic development. Moreover, it was possible to obtain reconstructed embryos that possess an apoptotic cell index indistinguishable from controls.

Nat. Chem. Biol. 10, 632–639 (2014); published online 22 June 2014; corrected after print 29 July 2014 In the version of this article initially published, Julia TCW's name was misspelled as Julia T C W. In addition, her initials in the author contribution statement should have read J.T. instead of J.T.

A separação entre massa interna celular (MCI) e trofectoderma (TE) é o primeiro evento de diferenciação celular que ocorre no embrião mamífero, seguida pela diferenciação na MCI do epiblasto (EPI) e endoderma primitivo (PE). Falhas nestes processos de desenvolvimento inicial podem ocasionar perdas embrionárias e consequentemente econômicas. A influência do sistema de produção embrionária nos primeiros eventos de diferenciação celular precisa ser mais elucidada, refletindo em índices que ainda podem ser melhorados na produção de embriões bovinos in vitro. O soro fetal bovino (SFB), apesar de seus efeitos deletérios sobre fatores epigenéticos, ainda é usado em sistemas de produção in vitro e pode influenciar esta diferenciação. Ainda, as técnicas para estudo dos embriões normalmente envolvem a destruição dos mesmos e não permitem acompanhar em tempo real a dinâmica de expressão dos genes relacionados à diferenciação em MCI e TE. Novas técnicas como CRISPR/Cas9 permitem alteração direcionada da sequência de DNA genômico, assim, a introdução direcionada de proteínas fluorescentes repórteres permitiria a visualização em tempo real da expressão de genes de interesse nos embriões. Diante disso, nossos objetivos foram: testar a influência do soro fetal bovino nas primeiras diferenciações e, realizar a inserção dirigida de uma proteína repórter fluorescente na região do gene SOX2 por recombinação homóloga. Ainda, comparamos a eficiência da inserção gênica com uso do sistema CRISPR em diferentes momentos do desenvolvimento embrionário, com diferentes horários pós inseminação e com diferentes concentrações de material injetado. No primeiro experimento observamos que a retirada do SFB do cultivo embrionário não causa alterações na alocação de células na MCI ou TE, quando se alia esta remoção à renovação de uma parte do meio de cultivo às 90 horas pós inseminação (hpi). A ausência de suplementação de SFB ou de meio de cultivo levou à diminuição das células do TE, entretanto não alterou o número de células do PE. No segundo experimento, nenhum embrião produzido apresentou fluorescência, portanto todos os blastocistos tiveram seu DNA extraído para genotipagem. Foi realizada uma amostragem, e de 34 embriões verificados, um apresentou banda correspondente ao local do inserto e também banda correspondente ao embrião controle (wild type), sugerindo mosaicismo. Ao final, observamos que as melhores probabilidades de inserção gênica nos embriões bovinos aconteceram quando a injeção dos componentes do sistema CRISPR foi realizada após 8 horas de FIV e com maiores concentrações. Em conclusão, pudemos remover o SFB do nosso sistema de produção de embriões e surpreendentemente o SFB não influenciou a diferenciação em PE. Ainda, são necessários maiores estudos a fim de se otimizar a ferramenta para a obtenção de knock-in em embriões bovinos, principalmente para que se obtenha taxas de maior sucesso e redução da probabilidade de ocorrência de mosaicismo nestes embriões. The separation between inner cell mass (ICM) and trophectoderm (TE) is the first cell differentiation event that occurs in the mammalian embryo, followed by differentiation into the epiblast MCI (EPI) and primitive endoderm (PE). Failures in these early development processes can lead to embryonic and subsequent economic losses. The influence of the embryo production system on the first cell differentiation events needs to be further elucidated, reflecting in rates that can still be improved in the production of in vitro bovine embryos. Fetal bovine serum (FBS), despite its deleterious effects on epigenetic factors, is still used in in vitro production systems and may influence this differentiation. Furthermore, the techniques for studying embryos usually involve their destruction and do not allow real-time monitoring of the expression dynamics of genes related to differentiation in ICM and TE. New techniques such as CRISPR/Cas9 allow targeted alteration of genomic DNA sequence, thus targeted introduction of fluorescent reporter proteins would allow real-time visualization of the expression of genes of interest in embryos. Therefore, our objectives were: to test the influence of fetal bovine serum in the first differentiations and to carry out the targeted insertion of a fluorescent reporter protein in the region of the SOX2 gene by homologous recombination. Furthermore, we compared the efficiency of gene insertion using the CRISPR system at different moments of embryonic development, with different times after insemination and with different concentrations of injected material. In the first experiment, we observed that FBS removal from the embryonic culture does not cause changes in cell allocation in the ICM or TE, when this removal is combined with the renewal of a part of the culture medium at 90 hours after insemination (hpi). The absence of FBS or culture medium supplementation led to a decrease in TE cells, however it did not change the number of PE cells. In the second experiment, no embryo showed a fluorescent signal, so all blastocysts had their DNA extracted for genotyping. We sampled 34 embryos and one presented a band corresponding to the insertion site and also a band corresponding to the control embryo (wild type), suggesting mosaicism. We observed that best chances of gene insertion occurred when the injection of components of the CRISPR system was performed after 8 hours of IVF and with higher concentrations. In conclusion, we were able to remove SFB from our embryo production system and surprisingly SFB did not influence differentiation into PE. Still, further studies are needed in order to optimize the tool for obtaining knock-in in bovine embryos, mainly to obtain higher success rates and reduce the probability of occurrence of mosaicism in these embryos.