RESUMO
Ao preservar o espermatozoide suíno no estado líquido ou criopreservado, os componentes do plasma seminal (PS) contidos nos ejaculados podem alterar a capacidade de fertilização desses gametas. O PS contém substâncias essenciais para a manutenção da viabilidade e fertilidade dos espermatozoides. No entanto, esses componentes podem ser deletérios dependendo da quantidade ou duração do tempo de contato entre a ejaculação e a remoção do PS durante o processamento do sêmen para a conservação na forma refrigerada ou congelada. Foram identificadas substâncias que prejudicam (principal proteína plasmática seminal PSPI) ou melhoram (espermadesina PSP-I) a capacidade de fertilização dos espermatozoides. Dependendo dos cachaços e dos procedimentos de colheita de sêmen, a remoção do PS pode ser benéfica antes da preservação no estado líquido ou criopreservado. Em alguns casos, o PS removido antes da congelação pode ser adicionado de volta ao diluente de descongelamento, com efeitos positivos no sêmen descongelado e na viabilidade do espermatozoide no trato reprodutivo da porca. Neste texto, há um foco nos diferentes efeitos de PS em amostras de sêmen refrigerado e criopreservado de suínos com ênfase em como PS modula a função e morfologia das células espermáticas antes, durante e após a preservação de forma refrigerada ou criopreservada.(AU)
When preserving sperm in the liquid or cryopreserved state, seminal plasma (SP) components within ejaculates can alter fertilizing capacity of these gametes. The SP contains substances essential for maintenance of sperm viability and fertility; however, these components can be deleterious depending on quantity, or duration of time before there is removal of SP from sperm in semen processing. Substances that impair (Major seminal plasma protein PSPI - boar) or improve (e.g., spermadhesin PSP-I - boar) sper- matozoa fertilizing capacity have been identified. Depending on individual males and semen collection procedures, SP removal may be beneficial before preservation in the liquid or cryopreserved state. In some cases, SP that is removed can be added back to thawing extender with there being positive effects in thawed sperm and for sperm viability in the female reproductive tract. In this review article, there is a focus on different effects of SP in samples of cooled and cryopreserved semen from boar with there being emphasis on how SP modulates the function and morphology of sperm cells before, during, and after preservation in the refrigerated or cryopreserved state.(AU)
Assuntos
Animais , Masculino , Sêmen/fisiologia , Preservação do Sêmen/veterinária , Suínos/fisiologia , Criopreservação/veterináriaRESUMO
In this study, we investigated the effects of melatonin supplementation in the culture medium and blastocoel fluid removal (BFR) before vitrification on the quality and viability of in vitro-derived bovine embryos. After fertilization, presumptive zygotes were assigned to one of the following treatments: control, in vitro standard culture (IVC) medium; IVC + M10-9, IVC medium supplemented 10-9 M melatonin; or IVC + M10-9 BFR, IVC medium supplemented with 10-9 M melatonin plus BFR on day 7 (D7) of culture. D7 blastocysts were vitrified by the Cryotop method and, after 5 mo of storage, were warmed and incubated for an additional 72 h. The re-expansion rate was evaluated after 2 and 24 h, and the hatching rate was evaluated after 24, 48, and 72 h. At 72 h, the total number of cells (TNC); number of apoptotic cells (NAC); and expression of genes related to oxidative stress (HSPA5), cell metabolism (SLC2A3), cell repair (MSH6), placentation (KRT8 and PLAC8), and implantation (FOSL1) were assessed in the blastocysts. Less than 30% of the control blastocysts re-expanded until 2 h, whereas more than 85% of the IVC + M10-9 and IVC + M10-9 BFR blastocysts re-expanded (P < 0.05). The hatching rate of IVC + M10-9 BFR blastocysts increased at all time points (P < 0.05), reaching 66.8% at 72 h of incubation. The TNC was similar among treatments (P > 0.05), regardless of vitrification/warming and re-cultivation. The NAC:TNC was smaller for melatonin-treated blastocysts (P < 0.05). BFR increased HSPA5 (P = 0.0118) expression and did not affect SLC2A3, MSH6, KRT8, and FOSL1 expression (P > 0.05). In conclusion, melatonin (10-9 M) supplementation in the culture medium and BFR on D7 of culture increased the hatching rate 24, 48, and 72 h after warming of the vitrified embryos, indicating an improvement in cryotolerance.
Assuntos
Melatonina , Animais , Blastocisto , Bovinos , Criopreservação/veterinária , Suplementos Nutricionais , Técnicas de Cultura Embrionária/veterinária , Feminino , Fertilização in vitro/veterinária , Melatonina/farmacologia , Gravidez , VitrificaçãoRESUMO
Diacylglycerol acyltransferase-1 (DGAT1) is one of the DGAT enzymes that catalyzes the final step in the synthesis of triacylglycerol, which is a major component of the lipid droplets in embryos. Intracellular lipids accumulated in embryos produced in vitro have been associated with reduced cryotolerance and quality. The objective of the present study was to investigate the influence of DGAT1 inhibition on embryo development, quality, and post-vitrification survival, in addition to expression profiles of selected lipid metabolism-regulating and oxidative stress genes. Bovine cumulus-oocyte complexes were matured and fertilized in vitro and were cultured in synthetic oviduct fluid (SOF) supplemented with 5% fetal calf serum (FCS) alone (Control) or with 1, 5, 10 or 50 µM DGAT1 inhibitor (A922500®; D1, D5, D10, and D50, respectively) or 0.1% dimethyl sulfoxide (CDMSO: vehicle for DGAT1 inhibitor dilution) from 54 h post-insemination until Day 8 post insemination. No differences were found in blastocyst yield on days 7 and 8 in Control, CDMSO, D10, and D50 groups. Embryos cultured with 10 or 50 µM DGAT1 inhibitor had greater mitochondrial activity (P < 0.01), and increased number of cells (P < 0.05), while the cytoplasmic lipid content was reduced (P < 0.01), the latter associated with altered expression profiles of selected genes regulating lipid metabolism or genes related with oxidative stress (transcript abundance increased for SLC2A1 and SLC2A5 and decreased for DGAT1 and GPX1). Importantly, the survival rate of blastocysts produced with 10 µM DGAT1 was higher than that of Control, CDMSO and D50 groups at 72 h after vitrification and warming (73.8 vs 57.1, 55.9 and 56.1%, respectively, P < 0.001). In conclusion, inhibition of DGAT1 synthesis in bovine embryos produced in vitro abrogates the negative effect of FCS by decreasing their lipid content, increasing mitochondria activity and improving embryo cryotolerance, as well as favoring the expression of lipid metabolism regulating and oxidative stress-related transcripts.
Assuntos
Diacilglicerol O-Aciltransferase , Técnicas de Cultura Embrionária , Animais , Blastocisto , Bovinos , Criopreservação/veterinária , Diacilglicerol O-Aciltransferase/genética , Técnicas de Cultura Embrionária/veterinária , Fertilização in vitro/veterinária , LipídeosRESUMO
l-carnitine is a potent antioxidant used for in vitro culture systems. Controversial results have been reported using l-carnitine in culture medium at different stages of in vitro bovine embryo production. Cumulus-oocyte complexes (n = 843) were in vitro-fertilized and cultured and added (treatment group) or not added (control group) with l-carnitine. At day three of culture, each group was subdivided into two subgroups receiving no l-carnitine (group 1), 3.8 mM l-carnitine added during in vitro maturation (group 2), 1.5 mM added during the in vitro culture (group 3), and 3.8 mM and 1.5 mM added during the maturation and culture, respectively (group 4). At day 8, blastocyst embryos were examined for mitochondrial activity, the presence of lipid droplets, total cell number, gene expression, and cryotolerance by vitrification. The data were analyzed with a one-way analysis of variance. l-carnitine added in the late in vitro culture significantly reduced mitochondrial activity and lipid content, and upregulated ifn-τ and ptgs2 gene expression compared to controls (p < 0.05). l-carnitine supplementation did not significantly affect the embryo rate production or survival rate after vitrification and warming (p > 0.05). l-carnitine supplementation significantly improved embryo potential to develop viable pregnancies in agreement with a study reporting improved pregnancy rates.
Assuntos
Antioxidantes/farmacologia , Carnitina/farmacologia , Técnicas de Maturação in Vitro de Oócitos , Animais , Antioxidantes/metabolismo , Blastocisto/efeitos dos fármacos , Blastocisto/metabolismo , Carnitina/metabolismo , Bovinos , Criopreservação , Ciclo-Oxigenase 2/genética , Feminino , Fertilização in vitro , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Oócitos/crescimento & desenvolvimento , Gravidez , VitrificaçãoRESUMO
Equilibration time (ET) is the period during which sperm cells are in contact with cooling/freezing media components at a temperature of 5⯰C, providing a proper osmotic balance between the intra- and extra-cellular milieu. The present study aimed to determine the ET (0, 2, and 4â¯h) that results in greater post-thaw sperm quality and functions. Based on the post-thaw sperm membrane integrity and motility ratios, 20 ejaculates collected from five boars were classified as having good (GFE, nâ¯=â¯5) or poor (PFE, nâ¯=â¯15) freezing capacity. Ratios of post-thaw sperm with intact plasma membrane and acrosome were similar between ET (0â¯h: 37.02 % ± 2.85 %; 2â¯h: 34.59 % ± 7.12 %; 4â¯h: 37.87 % ± 4.44 %) in GFE samples. In PFE, ratios of sperm with intact plasma membrane and acrosome at post-thaw were greater (Pâ¯<â¯0.05) after an ET of 2 h than after an ET of 0 h (2 h: 26.16 % ± 1.54 % and 0 h: 16.74 % ± 1.59 %). Also, ratios of post-thaw sperm with relatively lesser membrane lipids disorder were greater (Pâ¯<â¯0.05) after an ET of 2 h than for other ET in both GFE (2 h: 21.97 % ± 4.24 % and 0 h: 16.63 % ± 2.38 %) and PFE (2 h: 16.65 % ± 1.40 % and 0 h: 13.23 % ± 1.25 %) samples. In conclusion, an ET of 2 h results in greater sperm cryotolerance in both GFE and PFE samples, which suggests that modifying the freezing protocol lead to an increase post-thaw sperm function and survival.
Assuntos
Adaptação Fisiológica/fisiologia , Congelamento , Preservação do Sêmen , Espermatozoides , Suínos , Animais , Sobrevivência Celular , Criopreservação/métodos , Criopreservação/veterinária , Congelamento/efeitos adversos , Masculino , Lipídeos de Membrana/metabolismo , Distribuição Aleatória , Análise do Sêmen/métodos , Análise do Sêmen/veterinária , Preservação do Sêmen/métodos , Preservação do Sêmen/veterinária , Motilidade dos Espermatozoides , Temperatura , Fatores de TempoRESUMO
Short-term treatment of mammalian oocytes with different stressors induces stress tolerance of embryos derived from these oocytes. The aims of this study were to evaluate effects on embryo development when there was treatment of oocyte complexes (COCs) used to derive the embryos with hydrogen peroxide (H2O2).The COCs were not incubated with H2O2: control (0 µM), or were incubated with 25, 50, 75, or 100 µM concentrations of H2O2 for 1 h prior to in vitro fertilization, and presumptive zygotes were cultured until day 7. Blastocysts at day 7 of development derived from H2O2-treated (25 µM treatment concentration) COCs were vitrified. Percentage of embryos undergoing cleavage was not affected by any treatment, while percentage of embryos developing to the blastocyst stage was less when there was treatment of COCs with 100 µM of H2O2. Embryo quality was less when COCs used to derive blastocysts were treated with 50, 75, or 100 µM concentrations of H2O2. There were lesser relative abundances of some mRNA transcripts of interest in blastocysts when there was treatment of COCs with H2O2. After vitrification, there were no differences in embryo re-expansion and hatching rates compared with fresh and vitrified blastocysts of the control group and those derived from COCs treated with 25 µM H2O2. In conclusion, treatment of COCs used to derive blastocysts with H2O2 does not induce stress tolerance in vitrified embryos of cattle; however, the viability of these blastocysts is similar to those of the control group.
Assuntos
Bovinos/embriologia , Criopreservação , Peróxido de Hidrogênio/farmacologia , Estresse Oxidativo , Vitrificação/efeitos dos fármacos , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Criopreservação/métodos , Criopreservação/veterinária , Técnicas de Cultura Embrionária , Pesquisas com Embriões , Embrião de Mamíferos , Desenvolvimento Embrionário/efeitos dos fármacos , Desenvolvimento Embrionário/fisiologia , Feminino , Fertilização in vitro/veterinária , Técnicas de Maturação in Vitro de Oócitos/veterinária , Masculino , Oócitos/citologia , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Oxidantes/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Regulação para Cima/efeitos dos fármacosRESUMO
O sêmen suíno criopreservado possui emprego comercial limitado devido à perda de qualidade espermática, que ocorre durante o processo de congelação e de descongelação. A célula espermática suína possui peculiaridades que contribuem para esta alta sensibilidade às baixas temperaturas, tais como estrutura e composição da membrana plasmática. Além disso, estudos recentes apontam a relação da presença de proteínas a nível de membrana espermática e de plasma seminal, com a criotolerância das células espermáticas. Portanto, esta revisão faz uma abordagem sobre a íntima relação entre o perfil protéico do sêmen e a sua congelabilidade, característica do sêmen, que pode variar de animal para animal e até entre ejaculados de um mesmo varrão. Pesquisas têm identificados diversas proteínas, que aparecem em maiores concentrações no sêmen de alta congelabilidade. A técnica de proteômica apresenta um grande potencial ligado à reprodução, podendo ser utilizada na identificação de marcadores de fertilidade ou de ejaculados aptos ao processo de congelação/descongelação, evitando, assim, gastos, sejam eles de manutenção de reprodutores com sêmen de baixa qualidade para a congelação ou de processamento do sêmen para criopreservação.
Cryopreserved swine semen has limited commercial use due to the loss of sperm quality that occurs during the freezing and thawing processes. The porcine sperm cell has peculiarities that contribute to high sensitivity to low temperatures, such as the structure and composition of the plasma membrane. In addition, recent studies point to the relationship between the presence of proteins at the level of sperm membrane and seminal plasma with the cryotolerance of sperm cells. Therefore, this review addresses the intimate relationship between the protein profile of the semen and its freezability. The freezability of the semen can vary from animal to animal and even between ejaculations of the same boar. Research has identified several proteins that appear in higher concentrations in semen with high freezability. The proteomics technique has a great potential linked to reproduction and can be used to identify fertility markers or ejaculates suitable for the freezing / thawing process, thus avoiding expenses, be they for the maintenance of boar with low quality semen for freezing or semen processing for cryopreservation.
Assuntos
Animais , Análise do Sêmen/veterinária , Criopreservação , Preservação do Sêmen/veterinária , Proteômica , SuínosRESUMO
O sêmen suíno criopreservado possui emprego comercial limitado devido à perda de qualidade espermática, que ocorre durante o processo de congelação e de descongelação. A célula espermática suína possui peculiaridades que contribuem para esta alta sensibilidade às baixas temperaturas, tais como estrutura e composição da membrana plasmática. Além disso, estudos recentes apontam a relação da presença de proteínas a nível de membrana espermática e de plasma seminal, com a criotolerância das células espermáticas. Portanto, esta revisão faz uma abordagem sobre a íntima relação entre o perfil protéico do sêmen e a sua congelabilidade, característica do sêmen, que pode variar de animal para animal e até entre ejaculados de um mesmo varrão. Pesquisas têm identificados diversas proteínas, que aparecem em maiores concentrações no sêmen de alta congelabilidade. A técnica de proteômica apresenta um grande potencial ligado à reprodução, podendo ser utilizada na identificação de marcadores de fertilidade ou de ejaculados aptos ao processo de congelação/descongelação, evitando, assim, gastos, sejam eles de manutenção de reprodutores com sêmen de baixa qualidade para a congelação ou de processamento do sêmen para criopreservação.(AU)
Cryopreserved swine semen has limited commercial use due to the loss of sperm quality that occurs during the freezing and thawing processes. The porcine sperm cell has peculiarities that contribute to high sensitivity to low temperatures, such as the structure and composition of the plasma membrane. In addition, recent studies point to the relationship between the presence of proteins at the level of sperm membrane and seminal plasma with the cryotolerance of sperm cells. Therefore, this review addresses the intimate relationship between the protein profile of the semen and its freezability. The freezability of the semen can vary from animal to animal and even between ejaculations of the same boar. Research has identified several proteins that appear in higher concentrations in semen with high freezability. The proteomics technique has a great potential linked to reproduction and can be used to identify fertility markers or ejaculates suitable for the freezing / thawing process, thus avoiding expenses, be they for the maintenance of boar with low quality semen for freezing or semen processing for cryopreservation.(AU)
Assuntos
Animais , Proteômica , Análise do Sêmen/veterinária , Preservação do Sêmen/veterinária , Suínos , CriopreservaçãoRESUMO
Saccharomyces uvarum has been recovered from natural habitats and traditionally fermented beverages (apple chicha) in Patagonia. However, this species has never been obtained from industrially relevant beverages like wine or cider in the same region. In this work, different strains belonging to the cryotolerant species S. uvarum were recovered from spontaneous cider fermentations carried out at low temperature in Red Delicious apple must. The strain S. uvarum NPCC1420 obtained from this cider and selected for its physiological and technological features, evidenced a better adaptation to the cidermaking process than a previously selected strain obtained from a less industrialized product called apple chicha. Some differences, like a higher ethanol and sulphite tolerance, seemed to be associated with differential domestication pressures suffered by each different strain. Moreover, the most important fermentative features of the strain NPCC1420 were a higher competition capacity than the strain NPCC1314 in non-sterile apple must, as well as significantly higher amounts of glycerol, 2-phenylethanol and 2-phenylethyl acetate than the strain isolated from apple chicha.
Assuntos
Bebidas Alcoólicas/microbiologia , Fermentação , Sucos de Frutas e Vegetais/microbiologia , Malus , Saccharomyces/isolamento & purificação , Saccharomyces/metabolismo , Bebidas Alcoólicas/análise , Argentina , Fenômenos Químicos , Temperatura Baixa , Etanol/análise , Manipulação de Alimentos/métodos , Sucos de Frutas e Vegetais/análiseRESUMO
Abstract 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) (p<0.05). After vitrification, pregnancy rates were similar at 35 d between Bl (38.0) and Bx (47.6) (p>0.05). 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.
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) (p<0,05). Después de la vitrificación, las tasas de preñez a los 35 d fueron similares entre Bl (38,0) y Bx (47,6) (p>0,05). 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.
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) (p<0,05). Apos a vitrificação, as taxas de prenhez foram semelhantes aos 35 d entre Bl (38,0) e Bx (47,6) (p>0,05). 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.