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1.
Poult Sci ; 99(2): 1185-1198, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32029149

RESUMO

The aim of this study was to evaluate the effects of freezing diluents supplemented in three potential amines/amino acids, namely, antioxidant cysteamine (2-aminoethanethiol [AET]), ergothioneine (ERG), and serine (SER), in optimization of chicken sperm cryopreservation. The semen of 36 Pradu Hang Dum males, selected based on their motility vigor score, was frozen by a simple freezing method using nitrogen vapors and dimethylformamide (DMF). In a first experiment, a wide range of AET, ERG, and SER doses were tested. Semen quality was evaluated after incubation at 5°C or after cryopreservation in straws in the Blumberger Hahnen Sperma Verdünner (BHSV) diluent + DMF (6% v/v) with or without AET, ERG, or SER. The best targeted doses of AET, ERG, or SER were then selected for experiment 2 that was focused on cryopreserved semen. Frozen-thawed sperm quality was evaluated by different in vitro tests and by evaluation of fertility. Objective motility parameters were evaluated by computer-assisted sperm analysis. Membrane integrity, acrosome integrity, and mitochondria function were evaluated using appropriate dyes and flow cytometry. Lipid peroxide production was assessed by the thiobarbituric acid test (malondialdehyde production). Fertility obtained with frozen-thawed semen supplemented or not in AET, ERG, or SER was evaluated after artificial insemination of laying hens. ERG and AET decreased sperm lipid peroxidation and decreased fertility, even at low doses. The presence of 4 mmol of SER significantly decreased lipid peroxidation, increased the frozen-thawed sperm quality, and increased fertility after sperm cryopreservation (90% vs. control 84%, P < 0.05). In a third experiment, the use of 1 mmol of sucrose (the best result of our previous study) added to 4 mmol of SER-supplemented extender was tested. This addition allowed to the highest levels of fertility (93%). In conclusion, the addition of 4 mmol of SER in semen cryopreservation diluents decreases peroxidation and improves the efficiency of the process.


Assuntos
Antioxidantes/farmacologia , Galinhas/fisiologia , Criopreservação/veterinária , Crioprotetores/farmacologia , Espermatozoides/efeitos dos fármacos , Animais , Cisteamina/farmacologia , Ergotioneína/farmacologia , Fertilização/efeitos dos fármacos , Masculino , Análise do Sêmen/veterinária , Serina/farmacologia
2.
Arq. bras. med. vet. zootec. (Online) ; 72(1): 71-78, Jan.-Feb. 2020. tab
Artigo em Português | LILACS, VETINDEX | ID: biblio-1088920

RESUMO

Objetivou-se avaliar o efeito do ácido docosa-hexaenoico (DHA), associado ou não ao Trolox®, na refrigeração de sêmen de garanhões da raça Mangalarga Marchador. Foram refrigerados 10 ejaculados nos diluidores: D1) BotuSêmen® (BS; controle); D2) BS + 30ngmL-1 de DHA (BS30DHA); D3) BS30DHA + 40µM de Trolox® (BS30DHA40T); D4) BS + 50ngmL-1 de DHA (BS50DHA); D5) BS50DHA + 40µM de Trolox® (BS50DHA40T). Após 48 horas de refrigeração, foram avaliados os parâmetros de movimento espermático, a integridade estrutural e funcional da membrana plasmática e a longevidade espermática. Todos os diluidores testados preservaram, de forma semelhante, a motilidade, a linearidade, a retilinearidade, a amplitude do deslocamento lateral da cabeça, a frequência do batimento flagelar cruzado, o percentual de hiperativos e a integridade estrutural e funcional da membrana espermática (P>0,05). O diluidor BS50DHA foi superior ao BS30DHA40T em preservar a VCL e a VSL e foi superior ao BS30DHA40T e ao BS50DHA40T em preservar a VAP e o índice de oscilação (P<0,05). Conclui-se que o uso do Trolox® em diluidores utilizados para refrigeração de sêmen de garanhões contendo ácido docosa-hexaenoico, nas concentrações propostas, não é indicado por alterar parâmetros de movimento espermático considerados importantes para a fertilidade.(AU)


The objective of this study was to evaluate the effect of docosahexaenoic acid (DHA), associated or not with Trolox®, in extenders for cooling semen from Mangalarga Marchador stallions. Ten ejaculates were cooled in the following diluents: D1) BotuSemen® (BS; control); D2) BS + 30ngmL-1 DHA (BS30DHA); D3) BS30DHA + 40µM Trolox® (BS30DHA40T); D4) BS + 50ngmL-1 DHA (BS50DHA); D5) BS50DHA + 40µM Trolox® (BS50DHA40T). After 48 hours of refrigeration, the sperm movement, structural and functional integrity of the plasma membrane and sperm longevity were evaluated. All extenders tested similarly preserved motility, linearity, straightness of trajectory, amplitude of lateral head displacement, beat cross frequency, hyperactive, the structural and functional integrity of the sperm membrane (P> 0.05). The BS50DHA extender was superior to BS30DHA40T in preserving VCL and VSL and was superior to BS30DHA40T and BS50DHA40T in preserving VAP and oscillation index (P< 0.05). It is concluded that the use of Trolox® in extenders for cooling stallion sperm containing docosahexaenoic acid at the proposed concentrations is not indicated because it alters sperm movement parameters considered important for fertility.(AU)


Assuntos
Animais , Masculino , Preservação do Sêmen/métodos , Preservação do Sêmen/veterinária , Criopreservação/veterinária , Ácidos Docosa-Hexaenoicos/administração & dosagem , Cavalos , Ácidos Graxos , Antioxidantes
3.
Anim Reprod Sci ; 212: 106238, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31864488

RESUMO

Reactive oxygen species (ROS) and free radicals are one of the major detrimental factors that can negatively affect the quality of sperm during cryopreservation. Melatonin is an effective antioxidant and free radical scavenger in various cells. In this study, therefore, the aim was to evaluate the post-thawed quality of spermatozoa after cryopreservation of rooster semen in freezing extender supplemented with melatonin. Semen samples from seven Green-legged Partridge roosters were pooled and diluted with EK extender supplemented with 10-3, 10-6, or 10-9 M melatonin (control sample was prepared without supplementation with melatonin), and the pooled sample was subjected to cryopreservation. Post-thawed sperm motility was determined using the IVOS system, whereas plasma membrane status, acrosome integrity, mitochondrial activity, lipid peroxidation, chromatin status, and apoptotic-like changes were determined using fluorochromes and flow cytometry. Results, indicate post-thaw motile sperm cell count was greater (P < 0.05) in the frozen samples supplemented with melatonin (10-3 and 10-6 M) than the control sample. Although no significant differences were observed in post-thawed acrosomal integrity, plasma membrane integrity and mitochondrial activity were greater (P < 0.05) in samples frozen with melatonin (10-3 and 10-6 M) than that of the control sample. In addition, with supplementation of melatonin there was a decrease (P < 0.05) in the amount of lipid peroxidation, DNA fragmentation, and apoptotic-like changes after thawing. These results indicate there is a positive effect of melatonin supplementation in rooster semen freezing extenders on post-thaw sperm quality.


Assuntos
Criopreservação/veterinária , Crioprotetores/farmacologia , Melatonina/farmacologia , Preservação do Sêmen/veterinária , Espermatozoides/efeitos dos fármacos , Animais , Galinhas , Citometria de Fluxo/veterinária , Congelamento , Masculino , Análise do Sêmen/veterinária , Motilidade Espermática/efeitos dos fármacos
4.
Anim Reprod Sci ; 212: 106248, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31864493

RESUMO

The use of cryopreserved dolphin spermatozoa facilitates the exchange of genetic material between aquatic parks and makes spermatozoa accessible to laboratories for studies to further our understanding of marine mammal reproduction. Sperm cryopreservation in the bottlenose dolphin (Tursiops truncatus) has been developed for the exchange of gametes within the ex situ population. The aim of this study was to develop an effective method for refrigeration of bottlenose dolphin spermatozoa diluted in a commercial extender (BTS). In Experiment 1, the effect of temperature (5 compared with 15 °C) on sperm quality was evaluated during 7 days of storage at 100 × 106 spermatozoa/ml. In Experiment 2, the effect of the storage concentration (100 × 106 compared with 20 × 106 spermatozoa/ml) on sperm quality was assessed during 7 days of storage at 5 °C. In Experiment 1, total motility (including % of rapid sperm) was greater at 5 than 15 °C. When the effect of storage concentration was evaluated (Experiment 2), total motility and ALH were greater at the higher storage concentration (100 × 106 spermatozoa/ml). For both experiments, values for viability, acrosome integrity, and normal morphology variables were consistent throughout the 7 days of refrigeration. In Experiment 3, a microbiological study was performed to evaluate the effect of the refrigeration temperature and days of storage on bacterial growth. The results of microbiological analysis indicated there was Staphylococcus aureus in some samples, however, there was no effect of temperature or days of refrigeration. In conclusion, bottlenose dolphin semen can be refrigerated for a short to medium period of storage and there is maintenance of functionality of sperm when stored at 100 × 106 spermatozoa/ml at 5 °C.


Assuntos
Golfinho Nariz-de-Garrafa/fisiologia , Refrigeração , Preservação do Sêmen/veterinária , Espermatozoides/fisiologia , Animais , Temperatura Baixa , Criopreservação/veterinária , Masculino , Fatores de Tempo
5.
Anim Reprod Sci ; 212: 106254, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31864494

RESUMO

The purpose of this study was to assess the viability and growth of follicles in ovarian tissues of cattle vitrified using two non-permeating cryoprotectants (sucrose and trehalose) and two cryodevices (cryovial and cryotop). Cortical slices (1-2 mm3) from cattle ovaries (n = 5) were assigned to one of the 14 treatment groups. Cortical slices were vitrified in a TCM199 medium supplemented with ethylene glycol, DMSO, calf serum and either 0.5 M sucrose or trehalose, in cryovials or on cryotops. After warming, cortical slices were either fixed immediately for histology or grafted on a chorioallantoic membrane (CAM) of 10-day old chick embryos. Angiogenesis in ovarian tissues was determined. Viable and atretic preantral (primordial, primary and secondary) follicle densities were examined histologically. There was angiogenesis (chicken) in cortical slices grafted on the CAM by day 5 of culture, however, there was no difference for blood vessel densities when there was use of non-permeating cryoprotectants or cryodevices. Total, viable and atretic follicle densities did not differ (P > 0.05) with use of non-permeating cryoprotectants or cryodevices. The proportion of viable follicles was greater (P < 0.001) in fresh-control than CAM culture-control or vitrification groups. The inclusion of sucrose in the vitrification solution resulted in a larger number of atretic follicles than in the fresh-control group (P < 0.05). In summary, sucrose and trehalose, and cryotop and cryovial were equally suitable for vitrification of ovarian tissues of cattle. Vitrification of ovarian tissues of cattle with subsequent use of CAM culture adversely affected follicular development.


Assuntos
Bovinos , Membrana Corioalantoide/fisiologia , Criopreservação/veterinária , Folículo Ovariano/fisiologia , Preservação de Tecido/veterinária , Vitrificação , Animais , Embrião de Galinha , Feminino , Técnicas de Cultura de Tecidos , Preservação de Tecido/métodos
6.
Anim Reprod Sci ; 212: 106239, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31864495

RESUMO

The dromedary camel (DC) is a strategic animal for the exploitation of the desert and unhospitable lands (arid and semiarid areas). These animals are a genetic resource, locally adapted and more resilient to these environs, that may significantly contribute to food security and sustainable development of marginal land areas. Artificial insemination is the least invasive, least expensive technique for improving genetic selection and minimising transmission of venereal disease among animals and herds. Besides semen preservation protocols, specific approaches for the development of DC semen collection centres - biosecurity measures, screening for infectious diseases, management of animals, welfare, nutrition, control of seasonality, training, hygiene of semen collection and processing - have been considered less important aspects. The aim of this research is to describe the aspects related to the development of a DC semen collection centre, summarising the latest studies in the field of welfare, reproduction and diseases, and describing biosecurity and hygiene aspects related to semen collection and handling. Scientific gaps and requirements for maximising the production of good quality and safe-to-use semen doses with minimal risks of disease transmission are also described.


Assuntos
Camelus , Criopreservação/veterinária , Preservação do Sêmen/veterinária , Manejo de Espécimes/veterinária , Animais , Criopreservação/métodos , Criopreservação/normas , Inseminação Artificial/veterinária , Masculino , Estações do Ano , Preservação do Sêmen/normas , Manejo de Espécimes/normas
7.
Anim Reprod Sci ; 212: 106250, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31864497

RESUMO

Karan Fries is an Indian composite breed of cattle with superior milk production with a greater disease resistance than many other cattle. Understanding of the basic sperm and semen characteristics is necessary to develop the artificial insemination and sperm sexing methods. This study was conducted to evaluate the morphological, rheological and biochemical characteristics of Karan Fries (Indian composite breed) bull semen. Semen analysis was conducted using Computer Assisted Sperm Analysis (CASA) and rheological methods. Results from correlation assessments indicated there was a positive correlation among values for sperm velocity variables. Motility of bull sperm movements in a linear path were the important variables evaluated in this study. There was also characterization of the individual spermatozoa using scanning electron microscopy (SEM) and Raman spectroscopy. The sperm head length and width were 8.8 ±â€¯0.8 and 3.9 ±â€¯0.6 µm, respectively, and the approximate flagellum length was 59.4 ±â€¯4.5 µm. There was a difference in the Raman intensity among the individual spermatozoa. Results of this study could be important in designing and developing methods of artificial insemination, cryopreservation of semen and semen sexing.


Assuntos
Bovinos/genética , Bovinos/fisiologia , Análise do Sêmen/veterinária , Espermatozoides/fisiologia , Animais , Criopreservação/veterinária , Masculino , Preservação do Sêmen/veterinária , Motilidade Espermática
8.
Theriogenology ; 142: 390-399, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31708193

RESUMO

Cryopreservation of marine fish embryos causes to severe cryogenic damage, and to date, adults have not been reared from embryos that were cryopreserved. Here, we optimized vitrification factors to improve the survival and hatching rate of kelp grouper (Epinephelus moara) embryos after cryopreservation. We screened the effects of 11 vitrification solution concentrations (25-50%) on the survival rate of embryos at four developmental stages (16S, 18S, 22S, TB). We investigated the effects of different equilibration time (25-45min) on the survival rate and the influence of vitrification solutions on embryonic volume. In addition, we tested the effects of treating embryos at five different developmental stages (4-6S, 16S, 22S, TB, HB) with different vitrification solutions (35% PMG3S and 35% PMG3T), prechilling temperature (-5 °C and 4 °C) and prechilling time. In total, 9855 embryos were cryopreserved at 10 developmental stages, from optic capsule stage to pre-hatch stage. We found that kelp grouper embryos performed best at equilibration time of 30 min. Embryos at the tail-bud stage exhibited greater tolerance to vitrification than other stages. Vitrification solutions that contained sucrose showed better survival rates compared to embryos treated with vitrification solutions containing trehalose. Pre-chilling treatment improved viability before freezing, but did not improve viability after freezing. In the most optimal condition we identified in this study, the average survival, normal development and malformation rates of cryopreserved embryos were 6.32%, 2.36% and 3.49%, and 39.85% of the surviving embryos that were cryopreserved hatched. The hatched larvae gradually died at day 12 of cultivation, where the longest surviving individuals lived for 16 days. This study provides valuable data for improving survival and hatching rate of cryopreserved grouper embryos, and provides references for further exploring techniques in fish embryo cryopreservation.


Assuntos
Bass/embriologia , Criopreservação , Embrião não Mamífero , Vitrificação , Animais , Calibragem , Criopreservação/métodos , Criopreservação/normas , Criopreservação/veterinária , Crioprotetores/química , Crioprotetores/farmacologia , Desenvolvimento Embrionário/efeitos dos fármacos , Feminino , Masculino
9.
Theriogenology ; 142: 400-413, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31711689

RESUMO

Semen freezability is associated with genetic markers, and there is a diverse set of sperm transcripts that have been attributed to various cellular functions. RNA-Seq was performed to compare the transcript profiles of spermatozoa from boars with different semen freezability. We examined ejaculates from the Polish large white (PLW) boars that were classified as having good and poor semen freezability (GSF and PSF, respectively; n = 3 boars per group) by assessing post-thaw motility characteristics, mitochondrial membrane potential, plasma membrane and acrosome integrity. Total RNA was isolated from fresh spermatozoa from boars of the GSF and PSF groups and subjected to RNA-Seq (Illumina NextSeq 500 platform). Transcript abundance was assessed with the DESeq2, DESeq, and EdgeR Bioconductor R packages, and varying numbers of differentially expressed gene (DEG) transcripts were detected in the spermatozoa of each boar. Using RNA-Seq, we identified several genes associated with inflammation and apoptosis (FOS, NFATC3, ITGAL, EAF2 and ZDHHC14), spermatogenesis (FGF-14 and BAMBI), autophagy (RAB33B), protein phosphorylation (PTPRU and PTPN2) and energy metabolism (ND6 and ACADM) that were predominantly up-regulated in poor freezability ejaculates. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) validated the transcript expression levels detected by RNA-Seq and thus confirmed the reliability of this technique. Subsequent validation with western blotting showed that the expression of three proteins was in accordance with the transcript abundance. Overall, we demonstrated that the up-regulation of the DEG transcripts in spermatozoa was associated with poor semen freezability. We suggest that spermatozoa transcriptome profiling provides a foundation to further elucidate the relevance of sperm-related transcripts on cryo-survival. The sperm-related transcripts, namely FOS, NFATC3, EAF2, BAMBI, PTPRU, PTPN2, ND6 and ACADM, are potential markers for predicting the freezability of boar semen.


Assuntos
Congelamento , Perfilação da Expressão Gênica , RNA-Seq , Preservação do Sêmen , Espermatozoides/metabolismo , Suínos/genética , Animais , Criopreservação/métodos , Criopreservação/veterinária , Congelamento/efeitos adversos , Perfilação da Expressão Gênica/métodos , Perfilação da Expressão Gênica/veterinária , Masculino , RNA-Seq/veterinária , Análise do Sêmen/veterinária , Preservação do Sêmen/classificação , Preservação do Sêmen/normas , Preservação do Sêmen/veterinária , Espermatozoides/química , Suínos/metabolismo , Transcriptoma
10.
Theriogenology ; 142: 8-14, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31561132

RESUMO

The objectives of the present study were to analyze systematic effects on semen quality traits from fresh and frozen-thawed semen collected in and outside season. A total of 4,681 reports on semen traits of 121 stallions representing Arabian, Thoroughbred, Quarter Horse and four warmblood breeds used for artificial insemination at the Lower Saxon National stud Celle were edited for analysis of gel-free volume, sperm concentration, total number of sperm and total number of motile sperm in fresh semen and in frozen-thawed semen progressive motility, DNA fragmentation index and non-viable sperm. Month, year, age, breed and stallions effects were analyzed with a linear mixed model procedure. Breed differences were significant for sperm concentration, total number of sperm, total motile sperm number and DNA fragmentation index. Hanoverian stallions showed significant higher least squares means for sperm concentration, total number of sperm and total motile sperm number in fresh semen, whereas Thoroughbred had significant higher least squares means for DNA fragmentation index. Stallions with an age of 2-6 years had significant lower least squares means in sperm concentration, total number of sperm and total motile sperm number and progressively motile sperm post-thawing than the other age groups. Month was significant for all semen traits but progressively motile sperm post-thawing. Month by age class interaction showed a significant influence. Inter-stallion variance accounted for 27-71% and the two-way stallion by month interaction for 2-7% of the total variance of semen quality traits. The largest proportion of inter-individual variance among stallions was obtained for the DNA fragmentation index.


Assuntos
Criopreservação/veterinária , Congelamento , Cavalos/genética , Cavalos/fisiologia , Preservação do Sêmen/veterinária , Animais , Masculino
11.
Theriogenology ; 142: 15-25, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31574396

RESUMO

Although semen cryopreservation is an important, widely used technique for long-term sperm storage, it not only induces partially irreversible damages to sperm but might also deteriorate anatomical, biochemical, and structural organelles. These cellular and epigenetic modifications are the main reasons underlying the decline in sperm motility and fertility during the freeze-thaw process. Using the two Lake and Beltsville semen extenders, the present study aims to evaluate the epigenetic patterns (DNA methylation and histone modification), cellular parameters (e.g., membrane integrity, viability, DNA stability, mitochondria activity, and apoptosis status), and fertility potential of rooster semen collected from six mature roosters before and after cryopreservation according to a standard protocol. The results show that cryopreservation leads to significantly (P < 0.05) reduced values of the parameters examined when compared with those of fresh sperms. While the extenders used exhibit no difference with respect to DNA methylation (DNMT), the Lake extender leads to significant reductions (P < 0.05) in H3K9 acetylation (17.4 ±â€¯1.8) and H3K4 methylation (42 ±â€¯2.3) compared to the Beltsville (9.2 ±â€¯1.8 and 23 ±â€¯2.3, respectively). Compared to the Beltsville extender, the Lake one is also observed to yield a significantly (P < 0.05) superior sperm quality in terms of total motility (TM; 77.2 ±â€¯1.6 vs. 68.3 ±â€¯1.6), average path velocity (VAP; 71 ±â€¯1.4 vs. 53 ±â€¯1.4), and straight-line velocity (VSL; 52 ±â€¯1.5 vs. 34 ±â€¯1.5) as well as significantly (P < 0.05) higher viability (60 ±â€¯1.69 vs. 51 ±â€¯1.69) and membrane functionality (55 ±â€¯3.2 vs. 46 ±â€¯3.2). The Lake extender is also found to outperform the Beltsville one due to its significantly (P < 0.05) higher fertility rate (59.5% vs. 47.2%). The two extenders, however, exhibit no differences in DNA fragmentation, mitochondrial activity, or hatchability rate. The Beltsville extender showed to be superior to the Lake one due to its significantly greater reactive oxygen species percentage (ROS; 45.9 ±.3.2 vs. 28.5 ±â€¯3.2) and apoptosis (29 ±â€¯2.3 vs. 27 ±â€¯2.3). It may be concluded that the Lake extender is capable of improving the cellular and epigenetic parameters of rooster sperms during cryopreservation due to the crucial roles it plays in the protection of sperms against cryo-damages.


Assuntos
Galinhas/fisiologia , Criopreservação/veterinária , Epigênese Genética , Preservação do Sêmen/veterinária , Animais , Membrana Celular , Sobrevivência Celular , Crioprotetores , Fragmentação do DNA , Masculino
12.
Theriogenology ; 142: 131-137, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31593880

RESUMO

Endoplasmic reticulum (ER) stress, a dysfunction in protein folding capacity of the ER, is involved in many physiological responses including mammalian reproductive systems. Studies have shown that ER stress interferes with the developmental process of in vitro oocyte maturation and embryo development; however, little is known about its effects on bovine preimplantation embryonic development. In this study, we examined the effects of ER stress during IVC on developmental competency and cryo-tolerance in bovine embryos. IVF-derived zygotes were cultured in CR1aa medium supplemented with tauroursodeoxycholic acid (TUDCA) and/or tunicamycin (TM), which are ER stress-inhibitory and stress-inducing agents, respectively, for 8 days. TM treatment decreased the blastocyst developmental rate and increased the percentage of apoptotic cells compared to that in the control group (10.2 ±â€¯2.3% vs. 39.75 ±â€¯1.3% and 17.8 ±â€¯1.2% vs. 3.6 ±â€¯1.1%, respectively; P < 0.01). However, the blastocyst developmental rate was increased and the percentage of apoptotic cells was decreased by addition of TUDCA in IVC medium compared to that in the control group (50.9 ±â€¯0.9% vs. 39.75 ±â€¯1.3% and 1.13 ±â€¯1.0% vs. 3.6 ±â€¯1.1%, respectively; P < 0.01). Importantly, in the group treated with TM plus TUDCA, the developmental rate and the percentage of apoptotic cells in blastocysts were similar to that in the control group, indicating that TUDCA ameliorates the adverse effects of TM alone on embryo development. In addition, TUDCA treatment significantly reduced the reactive oxygen species, expression of ER stress (GRP78, ATF4, ATF6, IER1, and sXBP1) and pro-apoptotic (CHOP and BAX) genes, while it increased anti-apoptotic BCL2 gene expression and glutathione levels. Moreover, TUDCA improved blastocyst cryo-tolerance as marked by a significantly increased hatching rate and decreased the number of apoptotic cells recorded at 48 h after a post-warming. Therefore, in concordance with a previous report in mice or pig, we showed that TUDCA supplementation during IVC increases the developmental competency of bovine in vitro-derived embryos. Additionally, we found that the presence of TUDCA in IVC medium improves the cryo-tolerance of bovine embryos. These results suggest that modulation of ER stress during IVC contributes to the production of high-quality bovine embryos in terms of cryo-tolerance.


Assuntos
Bovinos/embriologia , Criopreservação/veterinária , Desenvolvimento Embrionário/fisiologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Ácido Tauroquenodesoxicólico/farmacologia , Tunicamicina/toxicidade , Animais , Antibacterianos/farmacologia , Colagogos e Coleréticos/administração & dosagem , Colagogos e Coleréticos/farmacologia , Relação Dose-Resposta a Droga , Técnicas de Cultura Embrionária , Ácido Tauroquenodesoxicólico/administração & dosagem
13.
Theriogenology ; 141: 219-227, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31084918

RESUMO

Male and female embryos are known to be different in developmental kinetics, metabolism, gene expression, and epigenetic patterns. Therefore, the objective of this study was to clarify whether the morphological criteria used to select embryos for cryopreservation lead to a deviation in the male:female ratio, and whether vitrification effects vary according to embryo sex. Initially, five sires were tested to evaluate the effect of the bull on embryo development, sex ratio, speed of development, and response to cryopreservation. Results showed that bulls affected (P < 0.05) embryo production, response to cryopreservation, and sex ratio. Then, one bull was selected, and used to produce embryos in vitro to characterize the responses of male and female embryos to vitrification. Results suggested that male and female embryos have the same morphological responses to vitrification, as no differences (P > 0.05) were observed between the two sexes in post-warming survival and re-expansion rates. However, their molecular responses as evaluated by gene expression (FOSL1, HSPB1, CASP3, CASP8, HSPA5, HSPA1A, G6PD, and PGK1) analysis indicated an effect of sex on vitrification; vitrified female embryos exhibited higher mRNA levels of HSPA1A, CASP3, and G6PD compared to their male counterparts. In conclusion, bulls affected embryo production, speed of development, sex ratio, and response to cryopreservation. Male and female embryos differed in their molecular responses to vitrification; and also, deviations in the male:female ratio when selecting embryos for cryopreservation were confirmed.


Assuntos
Blastocisto/fisiologia , Bovinos/embriologia , Criopreservação/veterinária , Técnicas de Cultura Embrionária/veterinária , Animais , Desenvolvimento Embrionário/fisiologia , Feminino , Masculino , Fatores Sexuais , Vitrificação
14.
Anim Reprod Sci ; 211: 106224, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31785625

RESUMO

The objectives of the present study were to evaluate the damage caused by cryopreservation on sperm DNA and estimate the percentage of cell apoptosis in tissue after thawing. Testicles of cats were sectioned into of 0.3 cm3 and 0.5 cm3 fragments and evaluated for DNA damage using acridine orange and semi-quantitatively through histo-morphological and immunohistochemical methods (caspase-3). Other fragments were placed in cryotubes with diluent containing either 3% glycerol or 3% propanediol, and were cryopreserved. Evaluation using acridine orange indicated there was a difference with use of propanediol and glycerol on DNA damage in 0.5 cm3fragments, with the latter being more effective than the former for cryopreservation. Results from histomorphological evaluations indicated there was a greater cell integrity among germ cells that were not cryopreserved, based on criteria assessed (detachment of cells from basal membrane, retraction of seminiferous tubule epithelium, visibility of the spermatogonia nucleoli and nuclear spermatogonia condensation), for both sizes of fragments. The values for these variables decreased after cryopreservation, with there being no differences as a result of size of fragment stored or between cryoprotectants used (P > 0.05). The staining for caspase-3 differed for the cytoplasm, nuclei and germ cells. Assessment of these staining patterns indicated the fresh fragments had an amount of cell damage and there was a similar amount of damage detected in cryopreserved fragments. This finding indicated that there was considerable efficacy in preserving the tissue fragments with use of the freezing protocols that were evaluated in this study.


Assuntos
Apoptose , Gatos , Criopreservação/veterinária , Fragmentação do DNA , Testículo/fisiologia , Preservação de Tecido/veterinária , Animais , Masculino , Espermatozoides/fisiologia , Preservação de Tecido/métodos
15.
Anim Reprod Sci ; 211: 106199, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31785628

RESUMO

Effects of different media and promoters on lipid peroxidation (LPO) in viable stallion sperm have not been reported. Aims of this study were to determine effects of three media (INRA-96™, Equipro CoolGuard™, and Biggers, Whitten and Whittingham [BWW]), and promoters (iron sulfate-Fe; ultraviolet light-UV; or control-no exposure to promoters) on viable sperm LPO using four different flow cytometric assays (i.e., BODIPY, Liperfluo, 4-hydroxylnonenal [4HNE], malonaldehyde [MDA]). Significant media x promoter interactions were detected using the Liperfluo, 4HNE, and MDA assays (P <  0.05); therefore, data were sorted by media and by promoters. With inclusion of milk-based media, there were similar concentrations of LPO in control samples with use of all LPO assays. The effect of iron, as a promoter of LPO production, was media dependent, and milk-based media protected sperm from iron-induced LPO production when there were assessments with all assays. In contrast, iron promoted LPO in sperm diluted in BWW when there was use of in all assays, except BODIPY, probably because of the different target molecule with use of this assay. Ultraviolet light was the most potent LPO promoter with all media and assays evaluated. Data indicate milk-based extenders are generally more LPO-protective than BWW early in the LPO production pathway (based on BODIPY and Liperfluo assays), but are less protective during the later stages of LPO production (based on 4HNE and MDA assays). The use of different media and promoters of LPO allowed for determination of early and late stages of LPO in viable stallion sperm.


Assuntos
Crioprotetores/farmacologia , Cavalos/fisiologia , Peroxidação de Lipídeos/efeitos dos fármacos , Espermatozoides/fisiologia , Animais , Criopreservação/veterinária , Meios de Cultura , Masculino , Espécies Reativas de Oxigênio , Preservação do Sêmen , Espermatozoides/efeitos dos fármacos , Espermatozoides/efeitos da radiação , Raios Ultravioleta
16.
Anim Reprod Sci ; 211: 106218, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31785630

RESUMO

In avian species, cryopreservation of semen is necessary for developing sperm cryobanks. It is very difficult, however to cryopreserve turkey sperm and have sperm be viable after thawing. Glycerol, the commonly used sperm cryoprotectant in many species, is toxic to sperm of avian species. The aim of this study was to evaluate whether the non-permeating dextran was effective for the cryopreservation and maintenance of turkey spermatozoa viability after thawing, avoiding the use of permeating cryoprotectants. Turkey sperm were diluted with a medium supplemented with 11% glycerol or dextran with a 1,000 molecular weight (MW), dextran with a 10,000 MW, or dextran with a 20,000 MW each at a 2%, 5%, or 10% concentration. Sperm kinetic characteristics, membrane and acrosome integrity (AI), and the capacity of spermatozoa to interact with the autologous perivitelline layer were evaluated after equilibration and cryopreservation. Results indicate that with use of glycerol and the 1,000 MW dextran there was lesser sperm viability after both equilibration and cryopreservation, compared with use of the 10,000 or 20,000 MW dextran compounds. There was a greater cryoprotective effect with the 10,000 and 20,000 MW dextran compounds at the 10% concentration with spermatozoa maintaining a greater functionality and capacity to interact with the autologous perivitelline layer. In conclusion, the results of this study indicate turkey spermatozoa could be effectively cryopreserved in extender without the use of glycerol as a penetrating cryoprotectant but with the use of the 10,000 or 20,000 MW dextran compounds at a 10% extender concentration.


Assuntos
Criopreservação/veterinária , Dextranos/farmacologia , Preservação do Sêmen/veterinária , Espermatozoides/fisiologia , Perus , Animais , Crioprotetores/farmacologia , Masculino , Motilidade Espermática/efeitos dos fármacos
17.
Anim Reprod Sci ; 211: 106205, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31785641

RESUMO

The aim of this study was to evaluate the effect of trehalose and lactose extenders on ejaculated and epididymal stallion sperm vitrification. Ejaculated semen samples were collected from seven fertile stallions, and cauda epididymis samples were collected from ten stallion carcasses after slaughter. Both the ejaculated and the epididymis samples were diluted and vitrified using INRA 96® and bovine serum albumin as well as trehalose or lactose. As a control, ejaculated and epididymal samples were collected and frozen using the conventional method. Vitrification was performed by immersing sperm suspensions directly in LN2. After thawing or devitrification, there was assessment of samples for sperm motility using computer-assisted analysis. Viability was assessed using SYBR-14 and propidium iodide (PI) and acrosome integrity by fluorescein using isothiocyanate combined with peanut agglutinin (FITC-PNA) and PI. Epididymal sperm vitrification with trehalose (EPT) or lactose (EPL) resulted in greater progressive sperm motility than sperm of the control sample (EPC). After post-thaw/devitrification of sperm in the EPT group, sperm motility was greater (P<0.001) compared to that using EPL (50.72 ± 5.09% compared with 34.21 ± 3.02%). The results from assessment of ejaculated sperm samples after undergoing the vitrification process indicated cells were less viable (P<0.001) than the control (EJC) sample. In conclusion, vitrification of epididymal stallion sperm using trehalose might be a beneficial alternative for the long-term storage of sperm samples with great economic value. Spermatozoa from vitrified ejaculates of stallions, however, had lesser motility and viability rates than samples subjected to conventional freezing.


Assuntos
Criopreservação/veterinária , Ejaculação/fisiologia , Epididimo/citologia , Cavalos/fisiologia , Preservação do Sêmen/veterinária , Vitrificação , Animais , Masculino , Análise do Sêmen
18.
Arq. bras. med. vet. zootec. (Online) ; 71(6): 1889-1895, Nov.-Dec. 2019. tab, graf
Artigo em Português | LILACS, VETINDEX | ID: biblio-1055129

RESUMO

A utilização da gema de ovo dificulta a padronização de meios diluidores e apresenta riscos biológicos. Assim, este estudo avaliou diferentes concentrações da lipoproteína de baixa densidade (LDL), em substituição à gema de ovo, para a confecção de diluentes para criopreservação espermática em ovinos. Foram utilizados um diluidor controle (CTR= 20% de gema de ovo) e cinco tratamentos, substituindo-se a gema pelas diferentes proporções de LDL (T1=6%; T2=8%; T3=12%; T4=16%; T5=20%), todos à base de TRIS-glicerol. Para o estudo, utilizaram-se dois ejaculados, de seis reprodutores da raça Santa Inês. Sessenta dias após a criopreservação, as amostras foram descongeladas e avaliadas subjetivamente quanto à motilidade total (MT, %) e progressiva (MP, %), ao vigor (1-5) e à integridade funcional (choque hisposmótico com água destilada, %) e estrutural (corante supravital eosina, %) das membranas espermáticas. As avaliações de vigor e funcionalidade de membrana não diferiram (P>0,05) entre os grupos. Entretanto, os grupos T4 (P<0,01) e T5 (P<0,05) foram superiores ao CTR para os parâmetros MT, MP e integridade estrutural de membrana, o que confirma que as LDLs podem ser alternativas eficientes para substituição da gema de ovo em diluidores para criopreservação de sêmen ovino.(AU)


The use of egg yolk makes it difficult to standardize extenders and presents biological hazards. Thus, this study evaluated different concentrations of low-density lipoprotein (LDL) to replace yolk extenders for production of sperm for cryopreservation in ovine. A control extender was used (CTR= 20% yolk) and five treatments, replacing the yolk by different ratios of LDL (T1= 6%; T2= 8%, T3= 12%; T4= 16%; T5= 20%) all based on TRIS-glycerol. For the study, two ejaculates from six Santa Ines breeding were used. Sixty days after cryopreservation, the samples were thawed and evaluated for total motility (MT, %) and progressive motility (MP, %), vigor (1-5) and the functional integrity (hyposmotic shock with distilled water, %) and structural (supravital dye eosin, %) of the sperm membranes. The evaluations of strength and membrane functionality didn't differ (P> 0.05) between groups. However, T4 (P< 0.01) and T5 (P< 0.05) groups were superior to the CTR for the MT, MP, and membrane structural integrity parameters, which confirms that LDLs can be efficient alternatives for yolk replacement in extenders for cryopreservation of ovine semen.(AU)


Assuntos
Animais , Masculino , Preservação do Sêmen/métodos , Ovinos , Criopreservação/veterinária , Lipoproteínas LDL/administração & dosagem
19.
Pol J Vet Sci ; 22(4): 661-666, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31867919

RESUMO

In this study, the effects of oleic (18:1 cis-9-octadecenoic acid) and linoleic (18:2 (n-6), 9,12-octadecadienoic acid) acids added to the embryo culture media for bovine embryonic development after vitrification were investigated in cattle. Following maturation and fertilization, the oocytes were placed in Charles Rosencrans (CR1aa) culture drops containing 10, 100, 500, and 1000 µM of oleic or linoleic acids. On day 7 or 8 of the culture, the blastocysts and expanded blastocysts were vitrified and warmed to evaluate the viability and development. High doses of oleic acid (1000 µM) in the culture media increased the viability of embryos after vitrification. Similarly, linoleic acid at 1000 µM increased the viability compared to the other linoleic acid doses. It was observed that the addition of essential fatty acids improved the development of embryos. Increasing the concentration of linoleic and oleic acid concentrations in the media proportionally advanced the embryonic development and hatching capability after vitrification/ /warming. Specifically, the addition of high doses of oleic acid had dramatic effects on the embryonic development after vitrification/warming probably due to the increased lipid storage. In conclusion, the present results suggest that the ratio of unsaturated fatty acids in the culture media affects significantly the embryonic development in vitro.


Assuntos
Bovinos/embriologia , Embrião de Mamíferos/efeitos dos fármacos , Ácido Linoleico/farmacologia , Ácido Oleico/farmacologia , Preservação de Tecido/veterinária , Vitrificação , Animais , Criopreservação/veterinária , Meios de Cultura , Técnicas de Cultura Embrionária/veterinária , Desenvolvimento Embrionário
20.
Cryobiology ; 91: 53-60, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31678072

RESUMO

The objective was to evaluate different permeating cryoprotectants to vitrify testicular tissue biopsies from adult collared peccaries. Five pairs of testicles were dissected into fragments (9 mm³) that were allocated to non-vitrified (control) and vitrified groups using a solid-surface method following exposure to different cryoprotectants (3.0 M dimethyl sulfoxide (DMSO), 3.0 M ethylene glycol (EG) or 1.5 M DMSO + 1.5 M EG). After warming, samples were evaluated for histomorphology, ultrastructure, viability, and proliferative capacity potential. The appropriate conservation of the ultrastructural organization of the seminiferous tubule in terms of lumen presence and cell junctions was only observed at the use of DMSO/EG combination. Regardless of the cryoprotectant, the vitrification effectively preserved cell nuclear visualization and condensation similarly as observed at the non-vitrified group. Moreover, DMSO/EG combination provided a better preservation of basal membranes of seminiferous tubules than DMSO (P < 0.05). The occurrence of cell swelling was more evident in the use of DMSO than EG (P < 0.05), but both isolate cryoprotectants were similar to the DMSO/EG combination. Only the DMSO/EG combination maintained the proliferative capacity potential for spermatogonia (3.69 NORs/cell) and Sertoli cell (3.19 NORs/cell) similar to controls (3.46 and 3.31 NORS/cell, respectively). Moreover, ~40% cell viability was found after vitrification independent of cryoprotectant. In conclusion, DMSO/EG in combination is better than DMSO or EG alone for SSV of testicular tissue biopsies from adult collared peccaries.


Assuntos
Criopreservação/veterinária , Crioprotetores/farmacologia , Dimetil Sulfóxido/farmacologia , Etilenoglicol/farmacologia , Testículo/citologia , Animais , Artiodáctilos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Criopreservação/métodos , Crioprotetores/química , Feminino , Masculino , Vitrificação
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