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Background and Aim: Oocyte in vitro maturation (IVM) is an appealing approach for several assisted reproductive technologies and dissecting oocyte maturation. Nonetheless, IVM leads to lower developmental competence and usually relies on undefined, serum-containing media. Therefore, biochemical profiling aimed to explore fluctuations in IVM media content during the acquisition of oocyte developmental competence. Materials and Methods: Bovine cumulus-oocyte complexes (COCs) underwent IVM in TCM199 medium with Earle's salts, supplemented with 2.0 mM L-glutamine, 10% fetal bovine serum, antibiotics, and 0.05 IU/mL porcine follicle-stimulating hormone (FSH+) or vehicle control (CTL) medium for 22 h. Results: FSH withdrawal (CTL) diminished several processes associated with the acquisition of oocyte developmental competence, such as reduced cumulus cell expansion, diminished estradiol synthesis (FSH+: 116.0±0.0 pg/mL vs. CTL: 97.6±18.0 pg/mL), and lower oocyte nuclear maturation rate (FSH+: 96.47% vs. CTL: 88.76%). Fresh media formulations (i.e., TCM199 with FSH or vehicle) were indistinguishable under biochemical profiling threshold conditions. Biochemical profiling showed similar total protein and lipid concentrations between groups. Further, total sugar concentrations diminished from fresh media to their post-IVM counterparts, albeit in an FSH-independent manner. Glycogen concentrations remained unaltered after IVM within CTL media, albeit were substantially lower after IVM under FSH+ conditions. Conclusion: FSH mediates the consumption of serum-derived glycogen by bovine COCs during IVM and implies that serum-free media should contain increased glucose concentrations to facilitate the acquisition of oocyte developmental competence.

Heat stress is an environmental factor that challenges livestock by disturbing animal homeostasis. Despite the broad detrimental effects of heat stress on reproductive function, the germline and the early preimplantation embryo are particularly prone. There is extensive evidence that elevated temperature reduces oocyte developmental competence through a series of cellular and molecular damages. Further research revealed that the oocyte respond to stress by activating cellular mechanisms such as heat shock response, unfolded protein response and autophagy to improve survival under heat shock. Such knowledge paved the way for the identification of thermoprotective molecules that alleviate heat-induced oocyte oxidative stress, organelle damage, and apoptosis. Therefore, this review depicts the deleterious effects of heat shock on oocyte developmental competence, heat-induced cellular and molecular changes, outlines pro-survival cellular mechanisms and explores thermoprotective molecules to improve oocyte competence.

Made available in DSpace on 2022-04-29T08:38:14Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-12-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Agencia Nacional de Promoción Científica y Tecnológica EPPIN (epididymal protease inhibitor) is a mammalian conserved sperm-binding protein displaying an N-terminal WFDC (whey-acidic protein four-disulfide core) and a C-terminal Kunitz protease inhibitor domains. EPPIN plays a key role in regulating sperm motility after ejaculation via interaction with the seminal plasma protein SEMG1 (semenogelin-1). EPPIN ligands targeting the SEMG1 binding site in the Kunitz domain are under development as male contraceptive drugs. Nevertheless, the relative contributions of EPPIN WFDC and Kunitz domains to sperm function remain obscure. Here, we evaluated the effects of antibodies targeting specific epitopes in EPPIN's WFDC (Q20E antibody, Gln20-Glu39 epitope) and Kunitz (S21C and F21C antibodies, Ser103-Cys123 and Phe90-C110 epitopes, respectively) domains on mouse sperm motility and fertilizing ability. Computer-assisted sperm analysis showed that sperm co-incubation with S21C antibody (but not F21C antibody) lowered progressive and hyperactivated motilities and impaired kinematic parameters describing progressive (straight-line velocity; VSL, average path velocity; VAP and straightness; STR) and vigorous sperm movements (curvilinear velocity; VCL, amplitude of lateral head movement; ALH, and linearity; LIN) compared with control. Conversely, Q20E antibody-induced milder inhibition of progressive motility and kinematic parameters (VAP, VCL and ALH). Sperm co-incubation with S21C or Q20E antibodies affected in vitro fertilization as revealed by reduced cleavage rates, albeit without changes in capacitation-induced tyrosine phosphorylation. In conclusion, we show that targeting specific epitopes in EPPIN Kunitz and WFDC domains inhibits sperm motility and capacitation-associated events, which decrease their fertilizing ability; nevertheless, similar observations in vivo remain to be demonstrated. Simultaneously targeting residues in S21C and Q20E epitopes is a promising approach for the rational design of EPPIN-based ligands with spermostatic activity. Department of Biophysics and Pharmacology Institute of Biosciences São Paulo State University, Botucatu-SP Department of Pharmacology Universidade Federal de São Paulo-Escola Paulista de Medicina, São Paulo-SP Instituto de Biología y Medicina Experimental Consejo Nacional de Investigaciones Científicas y Técnicas Department of Biological Sciences Universidade Federal de São Paulo - Campus Diadema, Diadema-SP Department of Biophysics and Pharmacology Institute of Biosciences São Paulo State University, Botucatu-SP FAPESP: 2015/08227-0 FAPESP: 2017/11363-8 FAPESP: 2019/13661-1 Agencia Nacional de Promoción Científica y Tecnológica: PICT

Background: Cryopreservation preserves cellular viability under low temperatures, resulting in diminished intracellular enzymatic activity and reduced cellular metabolism that ultimately allows preserving genetic material for indefinite periods of time. Embryos submitted to cryopreservation suffer from considerable morphological and functional damage, resulting in reduced survival and development rates. Objective: To evaluate pregnancy and delivery rates of in vitro-produced (IVP) Nellore (Bos indicus) embryos after vitrification under field conditions. Methods: The IVP embryos at blastocyst (Bl) and expanded blastocyst (Bx) were transferred fresh (n= 137) or after vitrification (n= 127). Results: Pregnancy rates at 35 d for fresh embryos were lower in Bl (41.6) than in Bx (60.9) (p0.05). Pregnancy loss at 60 d were similar (p>0.05) for both fresh (Bl: 3.1 and Bx: 4.8) and vitrified embryos (Bl: 1.9 and Bx: 4.7). Delivery rates were similar between groups (p>0.05). Conclusion: Both pregnancy and delivery rates of Bos indicus IVP embryos vitrified under field conditions are indistinguishable from fresh embryos.Key words: cattle reproduction, cryobiology, cryoinjury, cryotolerance, embryo surplus, embryo transfer. Resumen Antecedentes: La criopreservación se caracteriza por el mantenimiento de la viabilidad celular a bajas temperaturas, resultando en reducido metabolismo y actividad enzimática intracelular, lo que permite la preservación del material genético por períodos de tiempo indefinidos. Los embriones sometidos a ésta técnica sufren daños morfológicos y funcionales considerables, dando como resultado una sobrevivencia y tasas de desarrollo reducidas. Objetivo: Evaluar la tasa de preñez a partir de embriones Nelore (Bos indicus) producidos in vitro (IVP) después de la vitrificación bajo condiciones de campo. Métodos: Embriones IVP en los estadios de blastocisto (Bl) y blastocisto expandido (Bx) se transfirieron en fresco (n= 137) o después de la vitrificación (n= 127). Resultados: La tasa de preñez a los 35 d fue menor para los embriones transferidos en fresco en fase Bl (41,6) en relación con los Bx (60,9) (p0,05). Las pérdidas de preñez a los 60 d fueron similares (p>0,05) tanto para embriones en fresco en Bl (3,1) y Bx (4,8) como para los vitrificados (Bl: 1,9 y Bx: 4,7). Las tasas de nacimiento fueron similares entre los grupos (p>0,05). Conclusión: Las tasas de preñez y nacimiento de embriones IVP vitrificados de Nelore (Bos indicus) bajo condiciones de campo son semejantes a las de embriones en fresco.Palabras clave: criobiología, crioinjuria, criotolerancia, embriones sobrantes, reproducción de ganado, transferencia de embriones. Resumo Antecedentes: A criopreservação é caracterizada pela manutenção da viabilidade celular em baixas temperaturas, resultando em atividade enzimática intracelular e metabolismo celular reduzido, que permite a preservação do material genético por períodos indefinidos de tempo. Embriões submetidos à criopreservação sofrem danos morfológicos e funcionais consideráveis, resultando em sobrevivência reduzida e menores taxas de desenvolvimento. Objetivo: Avaliar a taxa de prenhez a partir de embriões Nelore (Bos indicus) produzidos in vitro (IVP) após a vitrificação sob condições de campo. Métodos: Embriões IVP nos estádios de blastocisto (Bl) e blastocisto expandido (Bx) foram transferidos a fresco (n= 137) ou depois da vitrificação (n= 127). Resultados: A taxa de prenhez aos 35 d foi menor para os embriões transferidos a fresco na fase de Bl (41,6), em relação aos Bx (60,9) (p0,05). As perdas de prenhez aos 60 d foram semelhantes (p>0,05) tanto para embriões a fresco nos estádios de Bl (3,1) e Bx (4,8), e vitrificados em Bl (1,9) e Bx (4,7). As taxas de nascimentos foram semelhantes entre os grupos (p>0,05). Conclusão: As taxas de prenhez e nascimentos dos embriões IVP vitrificados de Nelore (Bos indicus) sob condições de campo é semelhante àquela dos embriões a fresco.Palavras-chave: criobiologia, crioinjúria, criotolerância, embriões excedentes, reprodução de gado, transferência de embriões.

In the present study, we aimed to identify morphological and molecular changes of in vivo and in vitro-produced goat embryos submitted to cryopreservation. In vivo embryos were recovered by transcervical technique from superovulated goats, whereas in vitro produced embryos were produced from ovaries collected at a slaughterhouse. Embryos were frozen by two-steps slow freezing method, which is defined as freezing to -32 °C followed by transfer to liquid nitrogen. Morphological evaluation of embryos was carried out by assessing blastocoel re-expansion rate and the total number of blastomeres. The expression profile of candidate genes related to thermal and oxidative stress, apoptosis, epigenetic, and implantation control was measured using RT-qPCR based SYBR Green system. In silico analyses were performed to identify conserved genes in goat species and protein-protein interaction networks were created. In vivo-produced embryos showed greater blastocoel re-expansion and more blastomere cells (P 0.05). The expression level of CTP2 and HSP90 genes from in vitro cryopreserved embryos was higher than their in vivo counterparts. Unlikely, no significant difference was observed in the transcription level of SOD gene between groups. The high similarity of CPT2 and HSP90 proteins to their orthologs among mammals indicates that they share conserved functions. In summary, cryopreservation negatively affects the morphology and viability of goat embryos produced in vitro and changes the CPT2 and HSP90 gene expression likely in response to the in vitro production process.

SummaryHousekeeping genes (HKG) are paramount for accurate gene expression analysis during preimplantation development. Markedly, quantitative reverse transcription polymerase chain reaction (RT-qPCR) in ovine embryos currently lacks HKGs. Therefore, we tested 11 HKGs for RT-qPCR normalization during ovine parthenogenetic preimplantation development. Seven HKGs reached the qPCR efficiency threshold (97.20–105.96%), with correlation coefficients ranging from −0.922 to −0.998 and slopes from −3.22 to −3.59. GeNorm ranked glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and TATA-binding protein (TBP) as the best HKG pair, while H3 histone, family 3A (H3F3A) was the third HKG. Relative gene expression was measured for zinc finger protein X-linked (ZFX) and developmental pluripotency-associated 3 (DPPA3) transcripts during ovine parthenogenetic preimplantation development. ZFX did not show any transcript abundance fluctuation among oocytes, cleavage-stage embryos, and morulae. DPPA3 transcript abundance was also similar among all developmental stages, therefore suggesting that it may not display a maternal gene expression profile. In silico analysis of ovine DPPA3 mRNA and protein showed high conservation to bovine orthologues. However, DPPA3 orthologues differed in regulatory motifs. In conclusion, GAPDH, TBP and H3F3A are stable HKGs in ovine parthenogenetic embryos and allow accurate RT-qPCR-based gene expression analysis.

The work described here aimed to verify the efficiency of different extenders for cryopreservation of equine semen using sperm motility and acrosin activity as spermatic parameters. The semen was f...

The objective was to evaluate the role of ram effect combined with temporary weaning on P4, LH, follicular size and reproductive performance of hair ewes during postpartum. Pluriparous Santa Ines ewes (n = 99) during 15 to 30 days postpartum were selected and isolated from rams for an additional period of 30 days. Before exposure to rams, ewes were allocated to experimental groups, where on SI0, no suckling interruption was performed, and in SI24 and SI48, suckling interruption was performed for 24 and 48 hours, respectively. Mean P4 was according to anestrus condition before the introduction of rams, and it reached cyclicity levels after contact between genders. Ewes displaying a single estrus were more frequent in SI24 than SI48 (P 〈 0.05). The LH pre-ovulatory peaks were detected within 44 hours and 60 hours after introduction of rams, irrespectively of the group. Ovulatory follicles reached a more significant size in SI24 than SI0 and SI48 (P

Pluripotency-associated transcription factors (PATFs) are involved in several cellular processes, such as transcription initiation, cellular metabolism, and cell fate determination in preimplantation embryos. Despite growing understanding of PATFs in pluripotent human and mouse cells, their roles in other species or cell types remain limited. The work aimed to determine the transcript availability of OCT4 and four non-canonical PATFs (NAC1, NR0B1, RONIN, and ZFP281) in goat ovaries, eggs, and cumulus cells. Goat ovaries were collected at slaughterhouses and cumulus-oocyte complexes (COC) were retrieved from 3 to 6 mm follicles. The COCs were subject to in vitro maturation in TCM-199 medium containing 2 mM l-glutamine, 5.0 μg mL−1 FSH, 10% FBS, and antibiotics at 38.5 °C with saturated humidity and 5% CO2 during 24 h. Gene activity was detected by both conventional reverse transcription quantitative polymerase chain reaction (RT-PCR) and quantitative RT-PCR (RT-qPCR). In silico analysis were carried out for protein physical properties, phenetic analysis, and protein domains within goat, mouse, and human PATF orthologs. Products of all five PATFs analyzed (NAC1, NR0B1, OCT4, RONIN, and ZFP281) were detected in goat eggs, but NR0B1 was not detected in the ovary. Surprisingly, NR0B1, RONIN, and ZFP281 were also expressed in cumulus cells. All PATFs and their binding partners (i.e., OCT4-SOX2, RONIN HCF1, and NANOG-NR0B1-ZFP281-NAC1) showed similar protein size, molecular weight, and isoelectric point among species. The phenetic analysis clustered more closely goat and human proteins for OCT4, SOX2, NANOG, and NR0B1. Finally, both DNA-binding and transactivation domains were also highly conserved among species, particularly between goats and humans, suggesting that these PATFs may have conserved functions and similar target genes. In conclusion, non-canonical PATFs have distinctive transcript abundance in goat eggs and cumulus cells, while in silico approaches suggest that these genes may play similar roles to their mouse and human orthologs.

Pluripotency-associated transcription factors (PATF) contribute to cell fate decisions in embryos, cellular reprogramming, and derivation of stem cells. PATF hold species-specific mRNA fluctuations during early development but the regulatory determinants remain largely unknown in sheep. The aim was to prospect correlations between PATF gene expression patterns and mRNA regulatory features. The relative expression of PATF was investigated in sheep embryos by RT-qPCR and mRNAs were investigated for cis-regulatory elements and N6-methyladenosine (m6A) RNA methylation using bioinformatics. Four PATF gene expression patterns emerged with relatively distinct mRNA regulatory modes. RXRβ and TCF3 were detected in oocytes and mRNAs had variable 3’untranslated region (3’UTR) length. Three mRNAs (DPPA3, THAP11, and ZFX), which had similar abundance among developmental stages, frequently had short 3’UTRs and polyadelylation sites. NR5A2 and SP1 levels increased at the cleavage-stage but diminished in morulae, thus showing long 3’UTRs with multiple polyadelylation and m6A sites. Finally, mRNA abundance was low in oocytes and up-regulated in morulae (UTF1 and ZNF281) without a distinctive regulatory mode. Hence, sheep embryos hold PATF gene expression that correlates with mRNA 3’UTR length and the number of polyadenylation and m6A sites. These findings may lead to modulating sheep PATF to enhance reprogramming and stem cell derivation.

SummaryChemical oocyte enucleation holds the potential to ease somatic cell nuclear transfer (SCNT), although high enucleation rates remain limited to micromanipulation-based approaches. Therefore, this study aimed to test mitomycin C (MMC) for use in bovine functional chemical oocyte enucleation. Incubation of denuded eggs in 10 µg ml−1MMC for different periods did not affect most maturation rates (0.5 h: 85.78%A, 1.0 h: 72.77%B, 1.5 h: 83.87%A, and 2.0 h: 82.05%A) in comparison with non-treated controls (CTL; 85.77%A). Parthenogenetic development arrest by MMC was efficient at cleavage (CTL: 72.93%A, 0.5 h: 64.92%A,B, 1.0 h: 60.39%B,C, 1.5 h: 66.35%A,B, and 2.0 h: 53.84%C) and blastocyst stages (CTL: 33.94%A, 0.5 h: 7.58%B, 1.0 h: 2.47%C, 1.5 h: 0.46%C, and 2.0 h: 0.51%C). Blastocysts were obtained after nuclear transfer (NT) using MMC enucleation [NT(MMC): 4.54%B] but at lower rates than for the SCNT control [NT(CTL): 26.31%A]. The removal of the meiotic spindle after MMC incubation fully restored SCNT blastocyst development [NT(MMC+SR): 24.74%A]. Early pregnancies were obtained by the transfer of NT(MMC) and NT(MMC+SR) blastocysts to synchronized recipients. In conclusion, MMC leads to functional chemical oocyte enucleation during SCNT and further suggests its potential for application towards technical improvements.

SummaryThe addition of growth factors and vitamins enhances goat embryonic development in vitro. However, few attempts have been reported trying to identify supplementation regimens for oocyte maturation or embryo culture with additive properties. The present report was aimed to evaluate if retinoids [0.3 μM retinyl acetate (RAc) and 0.5 μM 9-cis-retinoic acid (RA)] supplementation during goat oocyte maturation and retinoids and/or 50 ng mL–1 IGF-I during embryo culture synergically enhanced embryonic development while diminishing the incidence of apoptosis. All combinations of RAc and RA treatment produced blastocysts with similar efficiencies, while IGF-I enhanced embryos yields irrespectively of retinoid addition. Moreover, retinoids and IGF-I supplementation showed similar caspase activity or DNA fragmentation indexes in blastocysts. In conclusion, supplementation with retinoids and IGF-I during goat embryo culture enhances blastocysts development without synergic reduction of apoptosis.

Heat stress (HS) is characterised by an elevation in body temperature that ultimately undermines organism physiology. Most livestock production occurs in tropical regions under potential HS conditions that diminish productive and reproductive potential. Despite extensive evidence of HS-mediated effects in cell function, stage-specific detrimental effects of HS during oogenesis remain elusive. Mouse models represent an attractive alternative for faster interrogation of stage-specific phenomena during oogenesis. Therefore, the aim of the study was to determine the effects of HS exposure during the major window of female mice germ-cell DNA methylation programming. CD1/Swiss female mice with litters (F0 progeny) at postnatal Day 10 (P10) were randomly allocated to HS (35°C/12-h light; 21°C/12-h dark) or control (CTL: 21°C/24 h) for 11 days. The F0 progeny were weaned at P21 and superovulated after reaching puberty at P35. F0 females were superovulated by intraperitoneal injections with 5.0 IU of equine chorionic gonadotrophin (PMSG) followed by 5.0 IU of human chorionic gonadotrophin (hCG) within a 48-h interval. Pre-implantation embryos were harvested at Day E3.5 in M2 medium under a stereomicroscope. One F0 female per litter was randomly mated to control mice when it reached 6 weeks of age. Data were subjected to least-squares analysis of variance using the General Linear Models procedure of SAS (SAS Institute Inc.). The experiment was replicated twice (CTL: n = 4 F0 females and HS: n = 4 F0 females). Preliminary results are given as LSM ± s.e.m. There was no effect of heat stress on the number of embryos collected per female (CTL: 9.75 ± 4.87 vs. HS: 11.25 ± 4.81) or the percentage of non-viable embryos (CTL: 25.0 ± 0.23% vs. HS: 42.5 ± 0.25%). However, heat stress tended (P = 0.07) to reduce the percentage of embryos that reached the morula stage from 63.5 ± 0.08% for CTL to 35.1 ± 0.09% for HS. The percentage of blastocysts collected (CTL: 11.45 ± 0.18% vs. HS: 22.32 ± 0.19%) and litter size of F0 females (CTL: 7.47 ± 1.76 vs. HS: 7.66 ± 1.47) was not affected by treatment. In conclusion, exposure of female mice to 11-day HS during the major wave of de novo DNA methylation during oocyte growth tended to reduce subsequent pre-implantation embryonic development, although it did not affect full-term development after natural mating.

Background: fertility rates using horse frozen-thawed semen remain lower than in other livestock species. This fact further suggests that horse semen hold intrinsic sensitivity to cryoinjury that must be investigated. Moreover, there is a substantial influence of genetic factors and diluent choice upon horse cryopreservation outcome. Collectively, these genetic and technical properties of horse semen could be explored to identify factors or conditions that may increase semen viability after freeze-thawing. The aim of this work was to evaluate the effect of diluents Botu-Crio®,Lactose-EDTA®, and INRA-82® on cryopreserved semen from stallions with high (HFA) and low freezability (LFA).Materials, Methods & Results: frozen-thawed semen was evaluated for motility, membrane integrity, and sperm DNA fragmentation using the thermoresistance test (TRT). Comparisons for each parameter were done in a pair-wise fashion between HFA and LFA semen at one-hour intervals during the TRT (0 h - 4 h). Sperm motility in HFA, regardless of the diluent, was larger (P < 0.05) than LFA, both on 0h and 1h. In the 2h evaluation, sperm motility using Botu-Crio® and Lactose-EDTA® was greater (P < 0.05) for HFA. Analysis of sperm membrane integrity was similar between HFA and LFA semen (P > 0.05) at 0 h and 3 h. Sperm DNA fragmentation was lower (P < 0.05) in HFA semen at 0 h and 1 h. Discussion: frozen-thawed semen from stallions of high freezing ability showed greater motility at all analysis, irrespectively of diluent choice, suggesting a strong influence of genetic factors on cryopreservation outcome. Membrane integrity was similar immediately after thawing but did differ later on other TRT time-points, irrespectively of diluent choice. As observed for motility, it was expected that sperm cells of stallions of HFA would show higher membrane integrity than their LFA counterparts. Sperm DNA fragmentation was quite low for both groups, as described in horses. Surprisingly, sperm DNA fragmentation incidence was constant throughout the analysis for both HFA and LFA. It was initially envisioned that increased DNA fragmentation would be found in semen from LFA stallions, since it is caused by multiple origins such as genetic factors. In conclusion, the semen diluent affects horse sperm motility after thawing, particularly from stallions with lower semen freezability.

Background: Proper timing for embryo collection and transfer in horses -which is critical for the success of this biotechnology- is still debated. Additionally, there is little information on this technology under tropical conditions. Objective: To determine the best day for collection and transfer of embryos in Mangalarga Marchador mares under Brazilian northeast’s conditions. Methods: Donors (n= 30) and recipients (n= 76) in diestrus phase were selected based on both clinical and gynecology examinations. Estrus was induced on both donor and recipient mares by intramuscular injection of 5 mg Dinoprost, aiming to obtain an ovulation interval of -1 to +3 between recipient and donor. Ovulation was induced with buserelin acetate when the largest follicle reached at least 35 mm in diameter. At this time, mares were subjected to artificial insemination at 48-hour intervals until ovulation. The embryos were collected on days 7, 8, and 9 after ovulation. Results: The embryo collection on day 8 was more efficient (p0.05) than day 9, which presented the same efficiency (p>0.05) as day 7. From a total of 76 embryos transferred to the recipients, that were between days 4 and 9 after ovulation, there was no influence (p>0.05) of the day of transfer on pregnancy rate. Conclusions: The embryo collection must be performed on day 8 after ovulation, and transfer can be performed on any day of that interval (4-9) without affecting the pregnancy rate. Resumen Antecedentes: El momento mas apropiado para la recolección y transferencia de embriones en equinos -que es fundamental para el éxito de esta biotecnología- continua siendo sujeto de estudio. Además, es escasa la información sobre esta tecnología en condiciones tropicales. Objetivo: Determinar el momento mas adecuado para la recolecta y transferencia de embriones en yeguas Mangalarga Marchador, en las condiciones del nordeste Brasileño. Métodos: Donadoras (n= 30) y receptoras (n= 76) en la fase de diestro se seleccionaron con base en los exámenes clínicos y ginecológicos. El estro de las yeguas donadoras y receptoras fue inducido con 5 mg de Dinoprost, vía intramuscular, intentando obtener un intervalo de ovulación de -1 a +3 entre la receptora y la donadora. La ovulación fue inducida con acetato de buserelina cuando el folículo mayor alcanzó 35 mm de diámetro. En ese momento, las yeguas fueron sometidas a inseminación artificial en intervalos de 48 horas hasta la ovulación. Los embriones fueron recolectados en los días 7, 8 y 9 después de la ovulación. Resultados: La recolecta de embriones en el día 8 fue más eficiente (p0,05) que en el día 9, el cuál presentó la misma eficiencia (p>0,05) que en el día 7. De un total de 76 embriones transferidos a las receptoras, que se encontraban entre el día 4 y 9 después de la ovulación, no se registró influencia (p>0,05) del día de la transferencia en la tasa de preñez. Conclusiones: La recolecta embrionaria debe ser realizada el día 8 después de la ovulación, y la transferencia puede ser realizada en cualquier día de este intervalo (4 a 9) sin que se afecte la tasa de preñez. Resumo Antecedentes: A importância do momentoda colheita e da transferência do embrião equino para o sucesso dessa biotécnica em equino continua sem ser completamente entendida. Adicionalmente, existe pouca informação sobre essa tecnologia em condições tropicais. Objetivo: Determinar o melhor dia para colheita e para transferência de embriões em eguas manga larga marchador nas condições do nordeste brasileiro. Métodos: Doadoras (n = 30) e receptoras (n = 76) na fase de diestro foram selecionadas com base nos exames clínico e ginecológicos. O estro das éguas doadoras e receptoras foi induzido com 5 mg de Dinoprost administrado por via intramuscular, buscando obter um intervalo de ovulação de -1 a +3 entre a receptora e a doadora. A ovulação foi induzida com acetato de buserelina quando o foliculo maior alcançou o tamanho de 35 mm de diâmetro. Nesse momento, as éguas foram submetidas a inseminação artificial em intervalos de 48 horas até a ovulação. Os embriões foram colhidos nos dias 7, 8 e 9 depois da ovulação. Resultados: A colheita de embriões no dia 8 foi mais eficiente (p0,05) do que o dia 9, o qual apresentou a mesma eficiência (p>0,05) que o dia 7. De um total de 76 embriões transferidos para as receptoras que se encontravam entre os dias 4 e 9 depois da ovulação, não se registrou influência (p>0,05) do dia da transferência sobre a taxa de prenhez. Conclusões: A colheita embrionária deve ser realizada no dia 8 depois da ovulação, e a transferência pode ser realizada em qualquer dia desse intervalo (4-9) sem que a taxa de prenhez seja afetada.

The work described here aimed to verify the efficiency of different extenders for cryopreservation of equine semen using sperm motility and acrosin activity as spermatic parameters. The semen was fractioned into two equal parts and resuspended in an 11% lactose solution in a 1:1 proportion, where it remained for 20 minutes at room temperature. The semen was centrifuged at 600

SummaryPluripotency-associated transcription factors (PATFs) modulate gene expression during early mammalian embryogenesis. Despite a strong understanding of PATFs during mouse embryogenesis, limited progress has been made in ruminants. This work aimed to describe the temporal expression of eight PATFs during both sheep and cattle preimplantation development. Transcript availability of PATFs was evaluated by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) in eggs, cleavage-stage embryos, morulae, and blastocysts. Transcripts of five genes were detected in all developmental stages of both species (KLF5, OCT4, RONIN, ZFP281, and ZFX). Furthermore, CMYC was detected in all cattle samples but was found from cleavage-stage onwards in sheep. In contrast, NR0B1 was detected in all sheep samples but was not detected in cattle morulae. GLIS1 displayed the most significant variation in temporal expression between species, as this PATF was only detected in cattle eggs and sheep cleavage-stage embryos and blastocysts.In silicoanalysis suggested that cattle and sheep PATFs share similar size, isometric point and molecular weight. A phenetic analysis showed two patterns of PATF clustering between cattle and sheep, among several mammalian species. In conclusion, the temporal expression of pluripotency-associated transcription factors differs between sheep and cattle, suggesting species-specific regulation during preimplantation development.

Background: Induction of ovulation is a key procedure for horse assisted reproduction technologies, such as for artificial insemination (AI) and embryo transfer. The application of hCG remains as the primary ovulation-inducing agent for horse assisted reproduction, but alternatives are in demand to avoid its adverse effects, such as loss of ovulation-inducing ef­ficiency over multiple applications by hCC-antibody production. Despite reports on alternative ovulation-inducing agents, pair-wise comparisons of such agents under similar conditions have been limited. Under such scenario, the work was aimed to determine the efficiency of both hCG and Buserelin at inducing ovulation in Mangalarga Marchador mares raised in the Northeast of Brazil under an AI program.Materials, Methods & Results: Mares were initially selected based on their reproductive performance, the absence of clinical-reproductive alterations and adequate body condition score. Mares in diestrus were randomly distributed in three experimental conditions, received 5 mg of Dinoprost and were monitored daily for estrus detection. After estrus detection, ovaries were monitored by ultrasonography, in 12-h intervals, until the follicle reached 35 mm. At this time-point, ovula­tion was induced with 0.042 mg of Buserelin endovenously, with 3,000 IU hCG by an intramuscular shot, and control mares received 2 mL of saline solution, also by an intramuscular shot. Both hCG and Buserelin displayed similar efficien­cies (P > 0.05) for induction of ovulation and that both agents were effective (P < 0.05) for such purpose, since greater percentages (P < 0.05) of induction on mares treated from those of the control. Moreover, the total number of ovulations in mares treated at the end of the experiment was not different (P > 0.05) from those found in the Control. All ovulations occurred within a 72-h period after treatment. It can be observed that in mares treated with hCG or Buserelin, ovulations occurred both in more mares (P < 0.05) and at earlier time-points than mares from the control. It is also possible to note that pregnancy was not different (P > 0.05) between hCG and Buserelin groups, and that pregnancy of mares treated with ovulation-inducing factors was similar to the control.Discussion: The majority of ovulations in mares occurred within initial 48-h after treatment for both hCG and GnRH, suggesting a similar potential for horse assisted reproduction. Both hCG and Buserelin are two commonly used agents for induction of ovulation in mares. As described here, the majority of ovulations occurred within initial 48-h after treatment, a fact which can be attributed to hCG and GnRH activity, since it can happen in intervals from 36 to 48-h after treatment. Pregnancy rates did not differ among groups. These results are under the working hypothesis that hCG and Buserelin would display similar efficiencies on pregnancy rates. Despite the understanding of hCG activity on induction of ovulation due to its high specificity toward LH receptors and results from a direct effect on diminishing estradiol concentration, increasing LH, and further inducing ovulation within 48-h after treatment. In contrast, Buserelin has a similar functional property but acts upon LH synthesis and its release. In conclusion, ovulation in mares can be induced with both hCG and Buserelin, and both ovulation-inducing agents do not affect pregnancy rates.Keywords: gonadotropin, releasing factor, ovary, follicle, corpus luteum.

The aim of this study was to evaluate the pregnancy rate of in vitro produced embryos of Gyr cattle ( Bos indicus ) after cryopreservation by the vitrification method under field conditions. Blastocysts in different developmental stages were transferred to recipient cows either fresh (n = 140) or warmed after vitrification (n = 138). The pregnancy rates obtained for fresh embryos were 46.15% (initial blastocyst), 46.93% (blastocyst) and 50.0% (expanded blastocyst) at 35 days post-fertilization, and 43.58% (initial blastocyst), 46.93% (blastocyst) and 50.0% (expanded blastocyst) at 60 days. The pregnancy rates after embryo vitrification were 35.0% (initial blastocyst), 42.3% (blastocyst) and 43.47% (expanded blastocyst) at 35 days post-fertilization, and 32.5% (initial blastocyst), 38.46% (blastocyst) and 43.47% (expanded blastocyst) at 60 days. Embryo vitrification or blastocyst development stage did not affect pregnancy rates or the incidence of embryonic death. In conclusion, vitrification of Gyr ( Bos indicus) embryos under field conditions is an efficient method that can be implemented to use surplus in vitro produced embryos without affecting pregnancy rates.

Introduction: During evolution, plants developed defense mechanisms against pathogen attack, including an infinity of molecular processes that are triggered by pathogen exposure, such as resistance gene synthesis (R). The NBS-LRR gene family is known as one of the most representative families in the R gene class, in which its protein domains are related to one form of plant defense mechanisms. Objective: To identify and characterize candidate sequences of the NBS-LRR gene family in the Vitis vinifera genome. Methodology: Initially, a seed sequence was selected from those curated in the NBS-LRR family and deposited in the UniProt database. This sequence was aligned via tBLASTn against the V. vinifera genome deposited in the NCBI, by adopting a cut-off of e-value ≥ e-10. The sequences were annotated, translated and had its conserved protein domains identified by both ORF Finder and CD-search tools, respectively. Finally, the prediction of the isoelectric point was performed, and the molecular weight was also estimated using the JVirGel 2.0 software. Moreover, the subcellular localization was carried out by the Cell-PLoc 2.0 software. Results and Discussion: A total of 40 candidate sequences were retrieved that are related to the gene of interest. The translated proteins showed a variation from 338 to 944 aa in size. A total of 33 complete NBS domains were found, from which 16 sequences had a whole RX-CC_like domain, while only two sequences have a full-length LRR domain. The candidate proteins had an isoelectric point from 5.25 to 9.46, a molecular weight varying from 38.47 and 108.14 kDa. All proteins showed cytoplasmatic subcellular localization, from which, 21 % also displayed an cellular membrane localization, in agreement with the data described in the literature. Conclusions: The results described here can contribute to a better understanding of the molecular characteristics of the NBS-LRR gene family and their role in defense mechanisms of grapevines to pathogens.

Univ Fed Rural Pernambuco, Dept Vet Med, Lab Biotecnol Aplicada Reprod Anim, BR-50010 Recife, PE, Brazil

The objective of the present study was to determine if separation distance between bucks and does during two distinct climate seasons could affect the reproductive performance of goats subjected to a 45-day mating season (MS). Anglo Nubian does (n = 120) were kept apart from bucks at distances of 2 m (T1), 300 m (T2), and 2000 m (T3) for 60 days prior to the 45-day MS during two distinct climate seasons [dry season (DS, February to March) and rainy season (RS, September to October)] in Sertânia, Pernambuco state, Brazil. There were no effects of distance of separation between bucks and does in any response variable evaluated. However, during the DS, the mean of the first estrous manifestation varied significantly (P>0.05) between groups [7.13±4.49 (T1), 8.84±5.64 (T2), and 6.37±4.21 (T3) days] and during the RS [7.33±5.74 (T1), 6.60±4.88 (T2) and 8.10±4.87 (T3) days]. Similar (P>0.05) estrous induction rates were found during both the DS [100.00% (T1), 100.00% (T2) and 95.50% (T3)] and the RS [100.00% (T1), 100.00% (T2) and 100.00% (T3)]. The estrous synchronization rate was found to be lower during the DS [36.60%; 30.00% (T1), 35.00% (T2) and 45.00% (T3)] than during the RS [56.60%; 50.00% (T1), 60.00% (T2) and 60.00% (T3)]. Pregnancy rates during the DS [P>0.05; 80.00% (T1), 70.00% (T2) and 75.00% (T3)] were lower than during the RS [P>0.05; 90.00% (T1), 90.00% (T2) and 95.00%(T3)]. In summary, the separation distance between bucks and does did not affect the reproductive outcome of Anglo Nubian goats over a 45-day MS under tropical conditions. Greater reproductive outcome was observed during the RS than the DS regardless of the separation distance between bucks and does.

This study aimed to evaluate the effect of temporary suckling interruption associated with the male effect on reproductive performance of pluriparous Santa Inês ewes. The females were kept apart from the males for 60 days and then randomly distributed into three treatments associated with the male effect (DT0, DT24 and DT48); in DT0, there was no suckling interruption; in DT24, suckling was interrupted for 24 hours, and in DT48, sucking was interrupted for 48 hours. Estrous distribution was observed within 31 (DT0), 27 (DT24) and 38 (DT48) days of the breeding season. Estrous synchronization up to the 5th day of the mating season was observed in 15% (DT0), 30% (DT24) and 25% (DT48) of the females, with no difference among treatments. Estrous percentages were 90% (DT0), 100% (DT24) and 100% (DT48), with no difference among treatments. Pregnancy rates were 38.4% (DT0), 60.0% (DT24) and 45.0% (DT48) with no difference among treatments. Prolificacy was 1.43 (DT0), 1.17 (DT24) and 1.22 (DT48) and did not differ between treatments. In conclusion, temporary suckling interruption associated with the male effect is efficient to induce estrous but not to synchronize estrous or increase the pregnancy rates and prolificacy of Santa Inês ewes during a 45-day breeding season.

Commercial application of reproductive biotechnologies such as multiple ovulation and embryo transfer depends on its overall efficiency. Sheep embryo transfer is gradually gaining wider adoption, but pregnancy rates after embryo transfer remain lower than those derived from natural mating for most breeds. The work was aimed to evaluate embryonic and fetal losses in Santa Inês ewes carrying twin pregnancies by natural mating or embryo transfer. Ewes were subjected to synchronized natural mating by ram effect or used as recipients for embryo transfer. Ewes diagnosed as carrying twin pregnancies at day 25 were used in the experiment (n = 42). Conceptus viability was monitored by ultrasonography on days 30, 35, 40, 45, 50, and 55 after conception. Conceptus loss was similar (P 0.05) within natural mating 11/42 (26.19%) and embryo transfer 14/42 (33.34%). However, overall embryonic loss (80.0%) was greater (P 0.05) than fetal loss (20.0%), with no difference within groups The results allow the conclusion that conceptus loss after embryo transfer is similar to natural mating and occurs predominantly during the embryonic stages.

The reprogramming of somatic cells to pluripotency using defined transcription factors holds great promise for biomedicine. However, human reprogramming remains inefficient and relies either on the use of the potentially dangerous oncogenes KLF4 and CMYC or the genetic inhibition of the tumor suppressor gene p53. We hypothesized that inhibition of signal transduction pathways that promote differentiation of the target somatic cells during development might relieve the requirement for non-core pluripotency factors during induced pluripotent stem cell (iPSC) reprogramming. Here, we show that inhibition of Notch greatly improves the efficiency of iPSC generation from mouse and human keratinocytes by suppressing p21 in a p53-independent manner and thereby enriching for undifferentiated cells capable of long-term self-renewal. Pharmacological inhibition of Notch enabled routine production of human iPSCs without KLF4 and CMYC while leaving p53 activity intact. Thus, restricting the development of somatic cells by altering intercellular communication enables the production of safer human iPSCs.

This study was aimed to compare different cryoprotectants for vitrification of sheep embryos produced in vivo. Blastocysts were obtained from superovulated Santa Ines ewes and randomized into three groups: conventional freezing using ethylene glycol (EG) (control group), vitrification with EG and dimethyl sulfoxide (DMSO vitrification), or vitrification with EG and dimethylformamide (DMF vitrification). All groups were analyzed for embryonic viability (propidium iodide staining), re-expansion rate after thawing (at morphological and ultrastructural levels) and pregnancy rate after embryo transfer (ET). Embryos of DMSO vitrification group showed lower cell viability (44.44%), compared to DMF group and control embryos (77.77% and 100%, respectively). The ultrastructural study showed similar cryopreservation damage among control and DMF embryos, and these were less damaged than DMSO vitrified embryos. Embryos vitrified with DMF had higher rates of re-expansion in vitro (53.33%) than DMSO (26.66%), and control (33.33%). After ET, similar pregnancy rates were obtained from all groups (DMF: 45%, DMSO: 30%, control: 40%). Collectively, DMF vitrification is more efficient than DMSO vitrification and is indistinguishable from conventional freezing of sheep embryos.

Quantitative reverse transcription PCR (RT-qPCR) remains as an accurate approach for gene expression analysis but requires labor-intensive validation of reference genes using species-specific primers. To ease such demand, the aim was to design and test a multi-species primer set to validate reference genes for inter-genus RT-qPCR gene expression analysis. Primers were designed for ten housekeeping genes using transcript sequences of various livestock species. All ten gene transcripts were detected by RT-PCR in Bos taurus (cattle), Bubalus bubalis (buffaloes), Capra hircus (goats), and Ovis aries (sheep) cDNA. Primer efficiency was attained for eight reference genes using B. taurus-O. aries fibroblast cDNA (95.54-98.39%). The RT-qPCR data normalization was carried out for B. taurus vs. O. aries relative gene expression using Bestkeeper, GeNorm, Norm-finder, Delta CT method, and RefFinder algorithms. Validation of inter-genus RT-qPCR showed up-regulation of TLR4 and ZFX gene transcripts in B. taurus fibroblasts, irrespectively of normalization conditions (two, three, or four reference genes). In silico search in mammalian transcriptomes showed that the multi-species primer set is expected to amplify transcripts of at least two distinct loci in 114 species, and 79 species would be covered by six or more primers. Hence, a multi-species primer set allows for inter-genus gene expression analysis between O. aries and B. taurus fibroblasts and further reveals species-specific gene transcript abundance of key transcription factors.

The objective of this study was to test the “male effect” on the reproductive performance of Anglo Nubian does (n = 180), aged between 24 and 60 months, under different male-to-female ratios (1:20 – T20, 1:30 – T30, and 1:40 – T40) and climatic conditions (dry season – DS, and rainy season – RS). Does were randomly distributed into three groups (T20, T30, and T40) and were isolated from bucks at a distance of 300 m for 60 days before the start of the experiments. The first manifestation of estrous during the DS occurred 6.83 ± 4.54 (T20), 6.72 ± 4.56 (T30) and 7.05 ± 5.23 (T40) days following the onset of the breeding season (P>0.05). In the RS, onset of estrous was observed 6.60 ± 4.74 (T20), 6.70 ± 4.43 (T30) and 7.46 ± 4.54 (T40) days after the beginning of the breeding season (P>0.05). Estrous induction in females during the DS occurred in 95% (T20), 80% (T30), and 75.5% (T40) of all females. During the RS, estrous detection reached 100% (T20), 100% (T30), and 97.5% (T40) of all females, with no difference between all RS and DS groups. Estrous synchronization during the DS occurred in 35.00% (T20), 36.66% (T30), and 32.50% (T40) of all females, for an average occurrence of 34.72%. During the RS, synchronization occurred in 65% (T20), 70% (T30) and 62.25% (T40) of all females, for an average occurrence of 65.75%; no difference was detected between the RS and the DS. Pregnancy rates in the DS groups were 65.0% (T20), 70.0% (T30), and 62.5% (T40), while pregnancy rates in the RS were 90.0% (T20), 86.6% (T30), and 95.0% (T40). No difference was observed for conception rates between any of the RS and DS groups. Prolificacy during the DS was 1.30 (T20), 1.30 (T30) and 1.35 (T40), while in the RS prolificacy was 1.29 (T20), 1.25 (T30) and 1.30 (T40). Thus, the male effect can be used effectively for goats under 1:20–1:40 male-to-female ratios in a 45-day mating season under varying climatic conditions.

Experiments were carried out to investigate the beneficial effects of retinyl acetate (RAc) and retinoic acid (RA) on goat oocyte maturation as well as the effects of insulin-like growth factor-I (IGF-I), RAc and RA during embryo culture under chemically defined conditions. In Experiment 1,in vitromaturation (IVM) was performed in a chemically defined basic maturation medium (bMM) supplemented with 0.3 μM RAc or 0.5 μM RA. Presumptive zygotes and embryos (2–4 cells) were cultured in droplets of potassium simplex optimised medium (KSOM); however, none of the embryos reached the blastocyst stage. In Experiment 2, oocytes were matured in bMM + RAc or bMM + RA. Presumptive zygotes and 2- to 4-cell embryos were placed in fresh KSOM droplets supplemented with RAc, RA, IGF-I, RAc+IGF-I or RA+IGF-I. In Experiment 1, addition of RAc and RA to bMM increased (P < 0.05) the proportion of 2- to 4-cell embryos reaching the morula stage as compared to the control. In Experiment 2, supplementation of embryo culture media with retinoids and IGF-I increased (P < 0.05) the proportion of 2- to 4-cell stage embryos developing to the morula and blastocyst stage. Our data demonstrate that goat embryo production in chemically defined media could be improved by exogenous RAc or RA and by the interaction between retinoids and IGF-I, and that goat embryos can be producedin vitrofrom oocytes following protocols similar to those currently used for cattle.

Cryopreservation of preimplantation embryos represents a major challenge due to their shape and relatively large cells. Embryo source and cryopreservation method are key factors to cryotolerance efficiency and few reports have investigated more promising protocols for goat embryos. The study was aimed to compare different cryopreservation methods for goat in vitro produced (IVP) embryos. Goat blastocysts were subjected to conventional freezing (CF), Dimethyl sulfoxide vitrification (DMSO-V) and Dimethylformamide vitrification (DMF-V). Cryopreserved blastocysts were assessed for re-expansion, cell viability and in vivo development rates. Blastocyst re-expansion after cryopreservation was similar between groups, but cell viability was lower for DMF-V (32%) than CF (68%) and DMSO-V (60%). Pregnancy and delivery rates were similar for CF (60% and 50%) and DMSO-V (50% and 45%) and higher then DMF-V (20% and 15%), respectively. Finally, kidding rates were also indistinguishable for CF (40%) and DMSO-V (35%), but higher then DMF-V (12.5%). In conclusion, conventional freezing and vitrification using DMSO have similar efficiencies for cryopreservation of goat IVP embryos and cryoprotectant for vitrification affects its outcome.

The reproductive performance of postpartum Santa Inês (SI) and Morada Nova (MN) ewes treated with insulin or progesterone hormones in association with ram effect was evaluated. Ewes from SI (n = 69) and MN (n = 69) breeds were randomly allocated to three groups of each breed (T1-ram effect only; T2-ram effect + insulin; T3-ram effect + progesterone). Progesterone concentrations (ηg/ml; mean ± SD) before and after introduction of rams (n = 6) were 0.51 ± 0.22 and 3.78 ± 0.68 (T1), 0.65 ± 0.21 and 3.77 ± 0.78 (T2) and 0.52 ± 0.21 and 3.84 ± 0.84 (T3) in SI ewes and 0.74 ± 0.19 and 3.71 ± 0.56 (T1), 0.70 ± 0.21 and 3.79 ± 0.75 (T2) and 0.81 ± 0.14 and 3.87 ± 0.80 (T3) in MN ewes, respectively. Thus, lower progesterone concentrations were found before the introduction of rams (p .05). After the introduction of rams, preovulatory peaks of LH (ηg/ml) occurred at 28 (T1), 44 (T2) and 48 (T3) hr in SI ewes and at 64 (T1), 40 (T2) and 44 (T3) hr in MN ewes. The mean number of ovulations was similar between groups (p .05), was 1.3 ± 0.51 (T1), 1.5 ± 0.54 (T2) and 1.6 ± 0.51 (T3) in SI ewes and 1.3 ± 0.51 (T1), 1.6 ± 0.51 (T2) and 1.6 ± 0.51 (T3) in MN ewes. In conclusion, the ram effect alone is effective at inducing and synchronizing oestrus in sheep under postpartum anoestrus, irrespective of hormone treatments.

Somatic cell nuclear transfer (SCNT) allows animal cloning but remains technically challenging. This study investigated limitations to functional oocyte enucleation by actinomycin D (AD) as a means of making SCNT easier to perform. Denuding oocytes or inhibiting transcription before AD treatment revealed that the toxicity of this compound during bovine oocyte maturation is mediated by cumulus cells. Exposure of denuded oocytes to higher concentrations of AD (5–20μgmL−1) and stepwise reductions of the incubation period (from 14.0 to 0.25h) led to complete inhibition of parthenogenetic development. Bovine SCNT using this improved AD enucleation protocol (NT(AD)) restored cleavage rates compared with rates in the parthenogenetic and SCNT controls (P(CTL) and NT(CTL) respectively). However, NT(AD) was associated with increased caspase-3 activity in cleavage stage embryos and did not recover blastocyst rates. The removal of AD-treated oocyte spindle before reconstruction (NT(AD+SR)) improved embryo development and reduced caspase-3 activity to levels similar to those in the P(CTL) and NT(CTL) groups. Furthermore, mid-term pregnancies were achieved using NT(AD+SR) blastocysts. In conclusion, improvements in AD functional enucleation for bovine SCNT circumvents most cellular roadblocks to early embryonic development and future investigations must focus on restoring blastocyst formation.

Exposure of caprine oocytes and embryos to retinoids enhances embryonic development, but the mechanisms governing this phenomenon have not been characterised. The aim of the present study was to evaluate if the incidence of apoptosis is affected by the addition of retinyl acetate (RAc) and 9-cis-retinoic acid (RA) during in vitro maturation (IVM) of caprine oocytes. Embryonic development was recorded on days 3 and 8 post-fertilisation, and apoptosis was measured by caspase activity and DNA fragmentation (TUNEL assay). Control zygotes had lower capacity to cleave and reach the blastocyst stage (24.45 ± 2.32 and 5.32 ± 0.81, respectively) than those of RAc- (29.96 ± 1.62 and 7.94 ± 0.93, respectively) and RA-treated groups (30.12 ± 1.51 and 7.36 ± 1.02, respectively). Oocytes and blastocysts positive for TUNEL assay were more frequent, respectively, in the controls (8.20 ± 0.78, 8.70 ± 1.05) than in RAc (5.60 ± 0.52, 4.80 ± 0.51) and RA (6.40 ± 0.69, 5.40 ± 0.69). Caspase activity did not differ between control oocytes (7.20 ± 0.91), RAc (6.60 ± 0.68) and RA (7.30 ± 0.67), but it was reduced in RAc- (5.05 ± 0.62) and RA-treated blastocysts (5.75 ± 0.22) compared to controls (8.35 ± 0.71). These results indicate that the addition of retinoids during IVM increases the developmental potential of goat embryos with a concomitant reduction in apoptosis rates.

Pluripotency-associated transcription factors (PATF) play significant roles during early embryogenesis and in embryonic stem (ES) cells, such as control of cell-cycle progression, modulation of cellular metabolism, and transcriptional control of differentiation-inducing factors. The review aims to describe the current understanding of how these PATFs contribute to the early embryo and the ES-cell phenotypes. By a selection of representative examples of such PATFs, their roles are described, and some interesting questions are presented concerning their activity in pluripotent cells which have yet to be addressed.

Embriões antes da implantação possuem células pluripotentes, ou seja, células que apresentam a capacidade de se diferenciar em todos os tecidos que compõem o feto. O controle da pluripotência em nível molecular é estabelecido por diversos fatores, entre eles, os genes relacionados à pluripotência (GRPs). Estes genes contribuem para inibição do processo de diferenciação celular e manutenção da viabilidade de células pluripotentes. No entanto, apesar do crescente conhecimento sobre as funções dos GRPs em camundongos e humanos, pouco se conhece sobre a expressão espaço-temporal e funções dos GRPs em outras espécies, incluindo ovinos. Evidências em bovinos, humanos e camundongos demonstram que GRPs podem apresentar mecanismos de ação diferentes entre as espécies. O objetivo da revisão foi analisar a atividade dos GRPs em ovinos através do perfil de expressão espaço-temporal e funções, bem como apresentar alternativas para acelerar o entendimento da pluripotência na espécie.

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.

The expression of a subset of transcription factors is enriched in early preimplantation embryos, which contributes to their cellular plasticity. RONIN, NANOG and its associated proteins are PluripotencyAssociated Transcription Factors (PATF) that control relevant downstream pathways in pluripotent stem cells, but their activity in early embryos remained less understood. The work was aimed to determine the expression of RONIN and four NANOG-associated PATFs in goat preimplantation embryos. Goat embryos were produced in vitro by parthenogenetic activation. Gene transcripts of cleavage-stage embryos were investigated by reverse transcriptase-polymerase chain reaction (RT-PCR), while blastocysts were analyzed by both RTPCR and quantitative RT-PCR (RT-qPCR) assays. Gene transcripts of ZFP281, NAC1, and NR0B1 were detected in cleavage-stage embryos, while RONIN and OCT4 were not found expressed. Detection in blastocysts by RT-PCR confirmed the activity of NR0B1, RONIN, and OCT4. Moreover, all five PATF were detected in blastocysts by RT-qPCR (ZFP281, NAC1, RONIN, OCT4, and NR0B1). In conclusion, RONIN and NANOG-associated proteins are active during goat parthenogenetic preimplantation development and hold stage-specific expression patterns.

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.

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.