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1.
An Acad Bras Cienc ; 93(3): e20191229, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34076181

RESUMO

To evaluate the addition of antioxidants in extenders on post-thaw bovine semen quality and in vitro embryo production efficiency. Six semen samples were collected from five Holstein bulls. In the experiment I, the samples were diluted with AndroMed® and Bovimix® and added antioxidants glutathione (1.5 and 2.5 mM) and melatonin (0.5 and 1.0 mM). In the experiment II, the best treatments obtained in experiment I were used for in vitro fecundation. Glutathione did not improve sperm viability. Melatonin had a negative effect on semen characteristics. Andromed® showed better results in sperm kinetics parameters. Bovimix® was more efficient in maintaining cell integrity parameters. Significant correlation was found between sperm kinetics parameters and between cell integrity parameters. For in vitro embryo production, after oocyte selection, maturation, fertilization and cultivation were performed using the four treatments previously evaluated. Andromed® was more efficient in the cleavage rate, no effect of the addition of glutathione. However, the addition of 2.5 mM glutathione in the Bovimix® improved the cleavage rate. There was a significant moderate correlation between cleavage rate and sperm kinetic characteristics. Glutathione did not improve sperm viability. Melatonin reduced the maintenance of sperm characteristics. Andromed® was more efficient in in vitro embryo production and no effect of glutathione was found in this extender. Addition of 2.5 mM glutathione in the Bovimix® extender provided a higher cleavage rate.


Assuntos
Análise do Sêmen , Preservação do Sêmen , Animais , Antioxidantes/farmacologia , Bovinos , Criopreservação/veterinária , Crioprotetores , Humanos , Masculino , Análise do Sêmen/veterinária , Preservação do Sêmen/veterinária , Motilidade Espermática , Espermatozoides
2.
Theriogenology ; 171: 21-29, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34000687

RESUMO

As a magical oligosaccharide, trehalose has been revealed to enhance the post-thaw quality of stock semen. However, information regarding the cryoprotective mechanism of trehalose during cryopreservation has not yet been determined. This study was designed to observe the effects of trehalose on the proteome of ram frozen spermatozoa by applying the isobaric tag for relative and absolute quantification (iTRAQ) strategy combined with parallel reaction monitoring (PRM). A total of 1269 proteins were identified. Among them, there were 21 differentially expressed proteins (DEPs), with 9 up-regulated proteins and 11 down-regulated proteins in spermatozoa frozen with trehalose. These DEPs were primarily located in nucleus, cytoplasm, and extracellular region. The Gene Ontology (GO) enrichment analysis demonstrated the involvement of the DEPs in signal transduction, ion binding, oxidoreductase activity, response to stress, and catabolic processes. Based on the STRING analysis, tight functional correlations were observed between 6-phosphogluconate dehydrogenase, fructose-bisphosphate aldolase A isoform 1, 14-3-3 protein epsilon, tyrosine-protein kinase Fer, and beta-hexosaminidase subunit alpha precursor. Furthermore, 10 DEPs were verified using PRM, confirming the accuracy of the iTRAQ data acquired in this study. In conclusion, trehalose can modify the protein profile of ram spermatozoa during cryopreservation, which may be associated with its cryoprotective effects. Additionally, trehalose may function on frozen spermatozoa through antioxidation, involvement in glycolysis, and increment of spermatozoa tolerance to various stresses.


Assuntos
Preservação do Sêmen , Trealose , Animais , Criopreservação/veterinária , Crioprotetores , Masculino , Preservação do Sêmen/veterinária , Ovinos , Motilidade Espermática , Espermatozoides , Trealose/farmacologia
3.
Poult Sci ; 100(6): 101093, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33965806

RESUMO

Chicken semen cryopreservation is a tool for programs of genetic diversity management and endangered breeds conservation. Due to physiological features, the fertility rates of cryopreserved poultry sperm are lower than mammal species. Thus, improvement of the semen cryopreservation methods is required. A first study was performed by a 2 × 2 factorial design consisting of 2 methods of adding the cryoprotectant [Direct or Diluted (mixed with extender medium)] and 2 cryoprotectants (glycerol and dimethylacetamide). Then sperm quality indicators were evaluated after freezing. A second study with a 2 × 2 design was conducted to evaluate the effectiveness of bovine serum albumin (BSA) on the optimization of 2 different extenders (Lake and Animal Sciences Group [ASG]). Viability and motility variables were evaluated before and after freezing. There was no significant difference in sperm viability and motility variables between Direct or Diluted methods. Supplementation of extenders with BSA improved most of the sperm motility variables in both extenders before and after freezing. Progressive sperm, non-progressive sperm before freezing, and all post-thaw sperm motility parameters, except amplitude of lateral head displacement and beat-cross frequency, were increased in BSA-supplemented extenders (P < 0.05), and BSA improved sperm viability in ASG extender after thawing (P < 0.05). After thawing, the interaction between extender and BSA (P < 0.05), eliminated the differences between the 2 BSA-supplemented media in curvilinear velocity, straight-line velocity, average path velocity, and amplitude of lateral head displacement which were higher in non-supplemented ASG extender than nonsupplemented Lake medium. In conclusion, the direct or diluted methods of adding glycerol or dimethylacetamide, did not significantly affect the post-thaw sperm characteristics. BSA positively affected most of the post-thaw sperm motility indicators regardless of the type of extender and resulted in significantly higher post-thaw sperm viability in ASG medium.


Assuntos
Preservação do Sêmen , Sêmen , Animais , Galinhas , Criopreservação/veterinária , Crioprotetores/farmacologia , Masculino , Preservação do Sêmen/veterinária , Soroalbumina Bovina , Motilidade Espermática , Espermatozoides
4.
Theriogenology ; 171: 44-54, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34023618

RESUMO

Vitrification and slow freezing are the two commonly used embryo cryopreservation methods. In most studies, vitrification of intact embryos has proven superior in several respects, including cell and embryo survival and pregnancy rate. However, there is a lack of data for comparing these two methods in in vitro produced (IVP) bovine blastocysts, which have been subjected to the retrieval of trophectoderm (TE) biopsy. Day 7 IVP blastocysts were pooled and randomized into four groups: 1) non-biopsy (NB), 2) biopsy (B), 3) biopsy-vitrification (BV), 4) biopsy-slow freeze (BSF). The blastocysts in the B, BV, and BSF groups were subjected to TE biopsy. For the B group, this was followed by 5 hours (h) incubation and subsequent scoring of the biopsy-survival (re-expansion) rate before processing for further analyses. For the BV and BSF groups, the biopsy procedure was followed by 2 h incubation, allowing for a quick re-expansion, after which the blastocysts were subjected to vitrification and slow freezing, respectively. After warming and thawing, respectively, they were then incubated for 5 h followed by scoring the cryo-survival (re-expansion) rates before processing for further analyses. These included quantification of ICM and TE cells, cleaved caspase-3- and TUNEL-positive cells, quantitative PCR on cellular stress markers (SOD1 and PRDX1), and ultrastructural analysis. The biopsy-survival rate in the B group was 94% (307/326). The cryo-survival rate in BV (86%, 138/161) was higher than that in BSF (57%, 81/142; P < 0.001). No differences were noted between the average ICM, TE, and total cell numbers of the groups. The percentages of cleaved caspase-3-positive cells were higher in BV vs. NB (P < 0.05), in BSF vs. NB (P < 0.001), and in BSF vs. B (P < 0.001). The percentages of TUNEL-positive cells were higher in BV vs. NB (P < 0.05) and in BSF vs. NB (P < 0.001). The levels of mRNA abundance for SOD1 and PRDX1 in B, BV, and BSF were not different from that in NB. The ultrastructural analysis of blastocysts in the BV and BSF groups showed distension of extracellular spaces and appearance of intracellular vacuoles in the ICM, distension of mitochondria, and disorganization of mitochondrial cristae in both ICM and TE, and weakened tight junctions between adjacent TE cells. In summary, our findings demonstrate that vitrification yields a higher cryo-survival rate than slow freezing in biopsied bovine IVP blastocysts. However, biopsy-vitrification and biopsy-slow-freeze values are comparable in terms of ICM, TE, and total blastocyst cell numbers, as well as cleaved caspase-3- and TUNEL-positive cell rates. Moreover, biopsy and cryopreservation performed alone had no effect on ICM, TE, total blastocyst cell numbers, or TUNEL-positive cell rates. Biopsy and vitrification performed alone had no effect on the cleaved caspase-3 positive cell rates, whereas slow freezing resulted in an increased rate. Furthermore, double traumatization with a combination of biopsy and cryopreservation, either vitrification or slow freezing, resulted in increased rates of cleaved caspase-3- and TUNEL-positive cells.


Assuntos
Técnicas de Cultura Embrionária , Vitrificação , Animais , Biópsia/veterinária , Blastocisto , Bovinos , Criopreservação/veterinária , Técnicas de Cultura Embrionária/veterinária , Feminino , Congelamento , Gravidez , Taxa de Sobrevida
5.
Res Vet Sci ; 136: 444-452, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33812287

RESUMO

Semen cryopreservation is routine in cattle, but the results of artificial insemination need improvement. A strategy to these aims is the supplementation of the freezing extender with novel antioxidants. This study aimed at testing the natural antioxidants curcumin and crocin as supplements to the commercial extender BIOXcell for freezing semen from 8 Holstein bulls. We tested curcumin at 0.05 and 0.1 mM (CU0.05, CU0.1) and crocin at 0.5 and 1.5 mM (CR0.5, CR1.5), with 0.5 mM reduced glutathione (GSH0.5) as reference, and a control (CTL, without supplementation). The samples were evaluated post-thawing and after 5 h at 38 °C by CASA for motility and flow cytometry for viability, apoptotic, capacitation, acrosomal status, cytoplasmic and mitochondrial reactive oxygen species (ROS) production, and chromatin status (SCSA). Control and GSH0.5 showed similar results, possibly because of the good protection from BIOXcell. CU0.05 and CU0.1 showed little effects but increased cytoplasmic ROS production and motility ALH. CR0.5 and CR1.5 decreased viability and increased apoptotic features significantly post-thawing and after the incubation, resulting in lower motility (significant after the incubation) but decreasing SCSA %HDS (loose chromatin). Whereas crocin at these concentrations seems incompatible with BIOXcell, maybe because of a prooxidant activity, curcumin use merits further research, considering the elevation of ROS with no significant negative effects.


Assuntos
Antioxidantes/farmacologia , Carotenoides/farmacologia , Criopreservação/veterinária , Curcumina/farmacologia , Preservação do Sêmen/veterinária , Sêmen/efeitos dos fármacos , Animais , Bovinos , Meios de Cultura , Glutationa/farmacologia , Masculino , Motilidade Espermática
6.
Theriogenology ; 167: 111-119, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33813051

RESUMO

The objective of the study was to evaluate the effects of different cryopreservation techniques including glycerol-based cryoprotectant combinations on the structure and viability of testicular tissues from adult collared peccaries. Tissue biopsies (3.0 mm³) from 5 different individuals were allocated to 10 different groups: fresh control; slow freezing (SF), conventional vitrification (CV), or solid-surface vitrification (SSV); each of them using three different combinations of cryoprotectants [dimethyl sulfoxide (DMSO) + ethylene glycol (EG); DMSO + Glycerol; and EG + Glycerol]. After thawing/warming, samples were evaluated for histomorphology, viability, proliferative capacity potential, and DNA integrity. Most effective preservation of testicular histomorphology was achieved using SF and CV with DMSO + EG. However, the use of glycerol-based cryoprotectant combinations increased the occurrence of tubular cell swelling, tubular cell loss and shrinkage from the basal membrane. Cell viability was comparable among cryopreservation methods and cryoprotectant combinations. Regarding cell proliferative capacity, the use of SF with EG + Glycerol and SSV with DMSO + Glycerol impaired the conservation of spermatogonia proliferative potential compared to other treatments. Moreover, CV with DMSO + EG was better than SF with EG + Glycerol for Sertoli cell proliferation potential. Regarding DNA integrity, less damage occurred when using SF with DMSO + EG while more fragmentations were observed when using CV with EG + Glycerol or DMSO + Glycerol as well as SSV with EG + Glycerol or DMSO + Glycerol. In sum, SF and CV appeared to be the most suitable methods for the cryopreservation of adult peccary testicular tissues. Additionally, the use of glycerol-based cryoprotectant combinations did not improve testicular tissues preservation with DMSO + EG being the most efficient option.


Assuntos
Artiodáctilos , Glicerol , Animais , Criopreservação/veterinária , Crioprotetores/farmacologia , Dimetil Sulfóxido/farmacologia , Etilenoglicol/farmacologia , Congelamento , Vitrificação
7.
Theriogenology ; 168: 66-74, 2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-33862426

RESUMO

With the progressive increase in the use of reproductive biotechnologies in the cattle industry, like artificial insemination and in vitro embryo production, the accurate determination of fertilizing competence of cryopreserved sperm samples is an essential issue. The routine methodology to assess bull sperm quality relies primarily on count, viability and motility of spermatozoa. However, these parameters do not tightly predict the reproductive success of samples. Therefore, identification of complementary markers of sperm functionality to strengthen the predictability of traditional spermogram is desirable to improve livestock reproduction practices. Previous results from our laboratory indicated that α5ß1 integrin plays a key role in bovine sperm function and mediates their interaction with the female reproductive tract. Thus, this study aimed to investigate whether the localization of α5ß1 held a correlation with fertilizing ability of bovine cryopreserved semen samples. Firstly, we assessed the quality of samples from six different bulls (A-F). We determined motility and viability of sperm samples after thawing and selection. Additionally, we measured the capacitation state of the samples by chlortetracycline (CTC) assay in the presence or absence of heparin, as an indicator of their responsiveness to a capacitating stimulus. Based on these assays, samples were classified being A the bull with the lowest quality and F the bull with the highest quality. Then, we studied the presence and localization of α5ß1 integrin. This protein showed a distribution pattern in the acrosomal (A), post-acrosomal (P) and acrosomal + post-acrosomal (A + P) regions with different localization percentages among the studied samples. Next, we determined the fertilizing ability of the samples in in vitro fertilization (IVF) assays and performed correlation analyses between IVF outcome and the routine spermogram parameters or α5ß1 integrin localization patterns. When the percentage of cells showing α5ß1 integrin was compared to fertilization rate, no correlation was observed. However, the presence of α5ß1 integrin in P and A + P regions (PA pattern), positively correlated with IVF rate (p < 0.05). These results suggest that while routine semen analyses failed to predict sperm reproductive competence, integrin localization in post-acrosomal region (PA pattern) showed a positive correlation with IVF outcome, thus posing an attractive marker to predict more accurately the reproductive performance of an individual.


Assuntos
Integrinas , Motilidade Espermática , Animais , Bovinos , Criopreservação/veterinária , Feminino , Fertilidade , Masculino , Espermatozoides
8.
Theriogenology ; 166: 112-123, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33735665

RESUMO

Single nucleotide polymorphisms (SNPs) in the 5'-flanking regulatory regions of genes could affect their expression levels. This is a follow-up study aimed to identify polymorphic variants in the 5'-flanking regulatory regions of genes expressed in boar spermatozoa, and to predict the interactions of such variants with transcription factors (TFs) on the gene promoter activity, using bioinformatics. Five and six boars were classified as having good and poor semen freezability (GSF and PSF, respectively) according to post-thaw (PT) assessment of sperm motility and membrane integrity characteristics. The 5'-flanking region sequences of the 14 genes (FOS, NFATC3, EAF2, FGF-14, BAMBI, RAB33B, CKS2, LARS2, SLC25A16, ACADM, CPT2, CCT3, DTD2 and CCDC85A) were PCR amplified and analyzed by Sanger sequencing method. A total of 32 polymorphic variants were identified in the 5'-flanking regions of the genes, including 4 insertion/deletion (indel) polymorphisms, and 8 unknown (novel) SNPs. Multiple sequence alignment analysis revealed a 26-bp indel variant in the 5'-flanking region of the LARS2 gene, which showed greater protein expression in spermatozoa from boars of the PSF group. It was found that 17 polymorphic variants, observed in the differentially expressed (DE) genes, showed significant allele frequency differences between the GSF and PSF groups. Polymorphic variants in the 5'-flanking regulatory regions of the genes contributed to the decrease or increase in the binding affinity for different testis-specific TFs, such as SMAD1, NF-1, FOXMI, RXRA, STAT4 and C/EBPß. This study provides more insights into the mechanisms responsible for variations in transcriptional activity in promoters of genes expressed in boar spermatozoa. The allelic variants are promising genetic markers for predicting the freezability of boar spermatozoa.


Assuntos
Preservação do Sêmen , Animais , Criopreservação/veterinária , Seguimentos , Masculino , Preservação do Sêmen/veterinária , Motilidade Espermática , Espermatozoides , Suínos/genética
9.
Theriogenology ; 167: 13-23, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33743504

RESUMO

As one of the most powerful natural antioxidants, astaxanthin (Ax) has begun to be applied to the field of reproductive biology. Here we used porcine oocyte as a model to explore how Ax improves the oocyte potential during in vitro maturation (IVM), and we also investigated the cytoprotective effects of Ax on the vitrified oocytes. Ax supplementation (final concentration of 2.5 µM) was subjected for immature oocytes during vitrification and subsequent IVM; fresh oocytes were also matured in vitro in the presence or absence of 2.5 µM Ax. Our results showed that Ax significantly increased the survival rate of vitrified oocytes, and promoted the blastocyst yield of both fresh and vitrified oocytes after parthenogenetic activation and somatic cell nuclear transfer. The oocytes treated with Ax displayed significantly lower reactive oxygen species generation and higher glutathione level. Vitrification of oocytes had no impact on caspase-3, cathepsin B and autophagic activities; Ax significantly decreased the cathepsin B activity in both fresh and vitrified oocytes. Moreover, the relative fluorescence intensity of lysosomes was significantly increased in vitrified oocytes, which was recovered by Ax treatment. The mitochondrial activity did not differ between fresh and vitrified oocytes, and was significantly enhanced in Ax-treated oocytes. Furthermore, Ax significantly restored the decreased expression of BMP15, ZAR1, POU5F1, GPX4 and LAMP2 genes in vitrified oocytes. Both fresh and vitrified oocytes treated with Ax showed significantly higher mRNA levels of GDF9, POU5F1, SOD2, NRF2 and ATG5. Taken together, this study provides new perspectives in understanding the mechanisms by which Ax improves the developmental competence of both fresh and vitrified porcine oocytes.


Assuntos
Antioxidantes , Vitrificação , Animais , Antioxidantes/farmacologia , Criopreservação/veterinária , Técnicas de Maturação in Vitro de Oócitos/veterinária , Oócitos , Suínos , Xantofilas/farmacologia
10.
Theriogenology ; 167: 24-31, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33743505

RESUMO

Sperm are redox-regulated cells, and deregulation of their redox status is considered to affect male fertility and to reduce their fertilizing ability following biotechnological procedures, such as cryopreservation. Cystine (CysS), after incorporation in sperm via SLC7A11 antiporter, has been demonstrated to increase intracellular GSH content, the most important non enzymatic antioxidant. This study was aimed at investigating the role of SLC7A11 antiporter on frozen-thawed stallion sperm ability to respond to in vitro capacitating environment after post-thaw incubation with CysS and/or Sulfasalazine (SS), a specific inhibitor of SLC7A11 antiporter. Viability, motility, immunolocalization of tyrosine phosphorylated proteins and the ability to bind to heterologous zonae pellucidae were evaluated. Thawed sperm from seven stallions (2 ejaculates/stallion) was washed and resuspended in Tyrodes media; each thawed ejaculate was divided in Control (CTR) and 3 samples supplemented with: 0.5 mM Cystine (CysS), 500 µM Sulfasalazine (SS) and 0.5 mM CysS + 500 µM SS (CysS + SS). After 1 h of incubation at 37 °C, samples were washed twice, resuspended in capacitating BWW medium and incubated at 38 °C under 5% CO2. After 30 and 60 min, sperm motility, viability and tyrosine phosphorylated protein immunolocalization, used as capacitation status index, were evaluated. After 30 min of capacitation, 4 × 105 sperm were co-incubated with denuded pig oocytes in capacitation medium for 30 min for the heterologous binding assay. None of the sperm parameters studied (motility, viability and tyrosine phosphorylation) showed any difference respective to control. The number of sperm bound per oocyte (mean ± SEM) tended to increase in CysS group (44.0 ± 12.3) respect CTR (40.8 ± 10.8) while decreased in SS group (32.4 ± 7.8) (p < 0.01). Moreover, CysS + SS group showed a lower binding rate (32.0 ± 10.0) compared to CysS (p < 0.001). Our results suggest that CysS supplementation of thawed stallion sperm can influence their ability to bind to heterologous zona pellucidae as the inhibition of CysS incorporation by SLC7A11 reduced the number of sperm bound per oocyte. This effect does not seem to be ascribed to a modification of sperm motility, membrane integrity and tyrosine phosphorylation.


Assuntos
Preservação do Sêmen , Animais , Antiporters , Criopreservação/veterinária , Cistina/metabolismo , Ácido Glutâmico , Cavalos , Masculino , Preservação do Sêmen/veterinária , Motilidade Espermática , Espermatozoides/metabolismo , Suínos
11.
Theriogenology ; 167: 77-84, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33774369

RESUMO

The high viscosity of Camelidae semen continues to present a major impediment for its application in assisted reproduction technology. The exposure of epididymal spermatozoa (ES) to seminal plasma (SP) may provide an approach to enhance the development of assisted reproductive techniques in these important domestic species. Since the sperm glycocalyx plays a key role in reproduction we aimed to evaluate whether SP exposure modifies the surface glycosylation patterns of cryopreserved dromedary ES. Epididymal sperm was collected through retrograde flushing of the cauda epididymidis that were obtained from orchidectomized mature dromedary bulls. The collected samples were then cryopreserved after dilution with a tris citrate clarified egg yolk extender, with and without the supplementation of 15% SP. Post-thaw carbohydrate surface profiles of both control and SP-treated spermatozoa were analyzed using 15 fluorescent lectins. Morpho-functional properties were also investigated via computer assisted sperm analysis. Lectin-binding analysis of the glycocalyx in control sperm revealed the presence of (1) N-glycans terminating with lactosamine (Con A, PHA-L, and RCA120), in both acrosomal and tail regions. Whilst (2) α2,3-/α2,6-linked sialic acids (MALII, SNA), and O-linked glycans terminating with a single N-acetylgalactosamine residue (Tn antigen) (HPA, SBA) along with galactoseß1,3N-acetylgalactosamine (T antigen) (PNA) were observed in the acrosomal cap. The expression of both N-acetylglucosamine (sWGA and GSA II) and terminalαgalactose (GSA I-B4) residues was also noted in the acrosomal cap region of control sperm. Compared with controls, SP treated samples displayed: 1) the appearance of bisected di-triantennary complex-type N-glycans (PHA-E), terminating with lactosamine, as well as an increase of O-glycans terminating with Tn and T antigens in both the acrosomal and tail regions; 2) an increase in glycans containing α2,6-linked sialic acid, N-acetylglucosamine, and αgalactose in the tail region. The cytoplasmic droplets of both control and seminal plasma-treated sperm bound Con A, PHA-E, PHA-L, RCA120, HPA, PNA, sWGA, GSA I-B4, and GSA II. These results indicate that SP treatment affects the glycan composition of the dromedary camel ES glycocalyx. More comprehensive studies are required in order to evaluate the fertilization capacity of SP-treated ES in order to facilitate its application in dromedary camel assisted reproduction technology.


Assuntos
Camelus , Sêmen , Animais , Bovinos , Criopreservação/veterinária , Epididimo , Masculino , Espermatozoides
12.
Poult Sci ; 100(5): 101058, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33743498

RESUMO

Sperm can be selected as a natural vector for the production of transgenic animals. Methyl-beta-cyclodextrin (MBCD) removes cholesterol from the phospholipid membrane of sperm and improves the efficiency of DNA uptake by sperm. In experiment 1, fresh sperm was treated with various concentrations of MBCD. The direct effects of MBCD on sperm parameters were monitored. In experiment 2, different concentrations of MBCD (0, 1, 2, and 4 mmol) were assessed for the transfection of genetically exogenous construction to rooster sperm. Washed semen was divided into 5 equal groups for the incubation and transfection with a pcDNA3.1+/hG-CSF vector (exogenous DNA) as follows; Treatment I-Control (washed semen without DNA); Treatment II-Control (washed semen with DNA); Treatment III-(washed semen incubated with DNA and 1 mmol MBCD); Treatment IV-(washed semen incubated with DNA and 2 mmol MBCD); and Treatment V-(washed semen incubated with DNA and 4 mmol MBCD). We demonstrated that rooster spermatozoa spontaneously can uptake exogenous DNA; this was assessed using exogenous DNA amplification (sperm genomic DNA used as a template for PCR reaction) after DNase I treatment. In addition, total motility (TM), progressive motility (PM), velocity parameters [curvilinear velocity (VCL), straight linear velocity (VSL), sperm track straightness (STR), linearity (LIN)], membrane integrity (MI), and membrane functionality were posttransfectionally evaluated. The concentrations of 1 and 2 mmol MBCD significantly (P < 0.05) improved the motion characteristics and membrane integrity of fresh sperm. The presence of hG-CSF in rooster sperm was detected by PCR and based on sperm analyses MBCD (1 mmol) improved the percentage of motility (98.9 ± 0.81), membrane functionality (64 ± 1.64), and MI (76.2 ± 1.65) after transfection when compared with the other groups (P < 0.05). For the production of transgenic chicken, hens were inseminated (AI) by transfected sperm treated with 1 and 0 mmol MBCD. A PCR analysis of the blood samples and dead embryo tissues of chicks did not reveal the transgene integration.


Assuntos
Preservação do Sêmen , Animais , Galinhas , Criopreservação/veterinária , DNA , Feminino , Masculino , Preservação do Sêmen/veterinária , Motilidade Espermática , Espermatozoides , beta-Ciclodextrinas
13.
Theriogenology ; 164: 58-64, 2021 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-33550092

RESUMO

Mammalian sperm is highly susceptible to reactive oxygen species (ROS) during the cryopreservation process. Astaxanthin (AST), a red pigment of the carotenoid family, is recognized as having a variety of beneficial biological activities and effects, including antioxidant, anticancer, anti-diabetic, and anti-inflammatory. The present study aimed to investigate whether the presence of AST protected boar sperm from ROS stress during cryopreservation. Boar sperm was diluted with a freezing medium supplemented with different concentrations of AST (0, 0.5, 1, 2, or 5 µM). The addition of AST, especially at a concentration of 2 µM, exerted positive effects on post-thaw sperm motility parameters. Meanwhile, sperm plasma membrane integrity and acrosome integrity of post-thaw sperm were significantly increased, while lipid peroxidation was inhibited in response to 2 µM AST treatment. Interestingly, compared to the control, supplementation with 2 µM AST increased unsaturated fatty acids (UFAs) levels and decreased saturated fatty acids (SFAs) content in post-thaw sperm, leading to a decreased ratio of SFAs/UFAs in the AST group. In conclusion, the addition of AST to freezing extenders inhibited lipid peroxidation and regulated fatty acid composition of the sperm membrane, improved post-thaw sperm quality, and had no adverse effect on boar sperm in vitro fertilization (IVF) capacity and potential for embryonic development. Our data provide a novel insight into understanding the mechanisms of AST concerning protecting boar sperm quality against ROS damage during cryopreservation.


Assuntos
Preservação do Sêmen , Animais , Membrana Celular , Criopreservação/veterinária , Crioprotetores/farmacologia , Fertilidade , Masculino , Preservação do Sêmen/veterinária , Motilidade Espermática , Espermatozoides , Suínos , Xantofilas
14.
Res Vet Sci ; 136: 111-118, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33607571

RESUMO

The purpose of this systematic review was to evaluate the potential benefits of antioxidant enrichment of semen extenders. These substances are used to combat oxidative stress during processing and conservation of rooster semen. A literature search was performed in June 2020 using the keywords rooster AND (semen OR spermatozoa OR sperm OR ejaculate OR ejaculation). This report followed the PRISMA (Preferred Reporting Items for Systematic Review and Meta-Analysis) guidelines. The PICO (population intervention comparison outcome) question was defined to compare roosters (Population) which had added antioxidants in the semen (Intervention) compared to the no-antioxidant group (Control); the outcome was semen quality (Outcome). Only articles investigating rooster cooled or frozen enriched semen with antioxidant extenders (Gallus Gallus domesticus) were selected by reading the title and abstract, totalizing 38 articles. After full text reading, we found that only 13 studies carried out sperm characteristics and fertility assays. To assess article quality, 15 items related to rooster breeding conditions, seminal collection methodology, and analyzed variables (seminal characteristics and fertility test) were established. There were positive effects of antioxidants on the preservation of seminal characteristics (motility, viability, membrane integrity, antioxidant activity, and lipid peroxidation) and on semen fertility after the conservation process. We conclude that the antioxidants reduce the oxidative stress and improve fertilizing capacity. The most used substances for cooled semen are glutathione, CoQ10, and l-carnitine; whereas for frozen semen, resveratrol, lycopene, and quercitin are most frequently used.


Assuntos
Antioxidantes/farmacologia , Criopreservação/veterinária , Crioprotetores/farmacologia , Preservação do Sêmen/veterinária , Sêmen/efeitos dos fármacos , Animais , Galinhas , Criopreservação/métodos , Fertilização/efeitos dos fármacos , Masculino , Estresse Oxidativo/efeitos dos fármacos , Análise do Sêmen/veterinária , Preservação do Sêmen/métodos
15.
Theriogenology ; 162: 42-48, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33444915

RESUMO

The aim of this study was determine the viability and developmental competence of equine oocytes after IVM and vitrification using the Rapid-I method, as part of an effort to develop an effective equine oocyte vitrification protocol. Equine oocytes were collected by scraping ovarian follicles of slaughtered mares. A total of 1052 ovaries were used in this study, from which 3135 oocytes were obtained. Of the 2853 oocytes retrieved, 2557 underwent in vitro maturation for approximately 36 h. After in vitro culture, 1202 oocytes (47%) had a first polar body. To evaluate the toxicity of the solutions (Experiment I), oocytes were exposed to vitrification media without cryopreservation. Of all the experimental groups evaluated, the best results were obtained for IVM oocytes exposed to EquiproVitKit media (IVM + TOX EquiVitKit), with a viability rate of 69.5%. In the Experiment II, oocytes, either freshly collected from the ovary or after in vitro maturation (IVM), were vitrified using either the EquiPro VitKit or an in-house medium containing 18% Ficoll, 40% ethylene glycol and 0.3 M sucrose. Oocytes were stained with fluorescein diacetate and ethidium bromide to evaluate viability. In vitro matured oocytes vitrified using EquiproVitKit media (IVM + VIT EquiVitKit) had a cryosurvival rate of 63%. In the last part of the study (Experiment III), vitrified IVM oocytes were activated by 7.5 µM ionomycin in TCM-199 for 5 min TCM 199 (5 min) combined with 2 mM 6-DMAP in TCM-99 with 10% FBS (4.5 h) or in vitro fertilized using ICSI. Development of potential embryos after activation in TCM-199 medium, showed a cleavage rate was 10.2%, compared to 22.5% of oocytes cultured in G1/G2 medium. ICSI of vitrified IVM oocytes resulted in 20% embryo development to the 16-cell stage, compared to 33.3% in the control. The vitrification of oocytes after IVM by Rapid-I method is a good way to preserve genetic material in horses.


Assuntos
Injeções de Esperma Intracitoplásmicas , Vitrificação , Animais , Criopreservação/veterinária , Feminino , Cavalos , Técnicas de Maturação in Vitro de Oócitos/veterinária , Oócitos , Partenogênese , Injeções de Esperma Intracitoplásmicas/veterinária
16.
Theriogenology ; 163: 31-42, 2021 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-33503549

RESUMO

This study was performed with the aim of evaluating the influence of static magnetic field (SMF) of 60 mT on mouse Cumulus Oocytes Complexes (COCs) vitrification. The COCs were vitrified in the presence (Vit_SMF+) and absence of SMF (Vit_SMF-). Along with these groups, non-vitrified or fresh COCS, which exposed (nVit_SMF+) and non-exposed (nVit_SMF-) to magnetic field, were also considered. Survival and viability rates and mitochondrial activity as well as ultrastructure of oocytes were examined by trypan blue Staining (TBS), Annexin-PI Staining, JC1 staining and transition electron microscopy, respectively. Following in vitro fertilization (IVF) and embryo development, gene expression was carried out through qRT-PCR at blastocyst (BL) stage. The survival rate in Vit_SMF+ and Vit_SMF- decreased meaningfully in comparison with nVit_SMF- (P < 0.05), but there was no significant difference between SMF+ and SMF- groups. The mitochondrial activity in Vit_SMF- was significantly reduced compared to the nVit_SMF- group (P < 0.05), however its value in Vit_SMF+ returned to the control level. Ultrastructural study demonstrated that SMF could protect the COCs from cryoinjuries and reduced damaged features in ooplasm of the vitrified oocytes. There was no significant difference in fertilization rate. Although, BL formation was the highest rate in the Vit_SMF+ group, it was just substantially higher than the non-vitrified groups (P < 0.05). The significant changes of Oct4, Cdx2 and Nanog genes expression due to vitrification (Vit_SMF-) or SMF (nVit_SMF+) treatments (P < 0.05) as compared to control (nVit_SMF-), returned to the natural level after using SMF in vitrified derived blastocysts (Vit_SMF+). Totally based on the results, it is clear that static magnetic field improves mitochondrial potential activity and ultrastructure of mouse vitrified COCs. In addition, SMF enhances the embryo cleavage rate to blastocyst stage and modulates pluripotency in blastocyst embryos derived from vitrified COCs.


Assuntos
Criopreservação , Vitrificação , Animais , Blastocisto , Criopreservação/veterinária , Fertilização In Vitro/veterinária , Campos Magnéticos , Camundongos , Oócitos
17.
Animal ; 15(1): 100097, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33516021

RESUMO

Sperm vitrification has been recently developed, but fertility trials have not been performed yet in equine species. In this study, a new warming technique for vitrified donkey semen was developed and the uterine inflammatory response and fertility were compared to conventional freezing. In Experiment 1, sperm was vitrified in straws and warmed in 3 ml of extender or in a water bath at: 37 °C/30 s; 43 °C/10 s; and 60 °C/5 s. Sperm motility, plasma and acrosome membranes and DNA integrity were compared between treatments. In Experiment 2, jennies were inseminated twice (500 × 106 sperm) in the uterine body either with vitrified or frozen semen (2 cycles/jenny). Pregnancy rates and the uterine inflammatory response (polymorphonuclear neutrophil concentration; PMN) were evaluated after artificial insemination (AI). No differences between warming in extender/water bath were found and 43 °C/10 s was better than lower temperatures in terms of total (53.8 ±â€¯13.2%) and progressive sperm motility (41.4 ±â€¯11.4%). No differences in PMN concentration (×103 PMN/ml) were found between vitrified (276.8 ±â€¯171.6) or frozen (309.7 ±â€¯250.7) semen after AI. However, PMN decreased faster (P < 0.05) using vitrified semen. Pregnancy rates were greater for vitrified (22%) than frozen semen (10%) but not statistically different. In conclusion, donkey sperm vitrified in straws could be directly warmed in a water bath at 43 °C/10 s, reducing the uterine inflammatory response obtained after AI and promoting positive pregnancy outcomes. These findings confirm the possibility to use vitrified semen as an alternative for AI in jennies.


Assuntos
Preservação do Sêmen , Sêmen , Animais , Criopreservação/veterinária , Equidae , Feminino , Cavalos , Inseminação Artificial/veterinária , Masculino , Gravidez , Preservação do Sêmen/veterinária , Motilidade Espermática , Espermatozoides
18.
Res Vet Sci ; 135: 20-26, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33418187

RESUMO

Several vitrification protocols have been established for porcine oocytes so as to facilitate gene banking of female germplasm. Although live piglets have been successfully produced from pig oocytes vitrified at the germinal vesicle (GV) stage, the competence of vitrified oocytes to develop into the blastocyst stage is greatly compromised following cryopreservation. The focus of this review is to elucidate the impact of cryopreservation on the redox balance of pig oocytes, making special reference to the relevance of non-enzymatic and enzymatic antioxidant defences. Besides, the regulation of gene expression in response to oxidative stress is also considered. Finally, we discuss the effects of supplementing maturation and vitrification media with the exogenous non-enzymatic antioxidants that have hitherto yielded the most relevant results.


Assuntos
Criopreservação/veterinária , Oócitos/fisiologia , Estresse Oxidativo/efeitos dos fármacos , Suínos/fisiologia , Animais , Criopreservação/métodos , Feminino , Vitrificação
19.
Trop Anim Health Prod ; 53(1): 103, 2021 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-33417110

RESUMO

To investigate the effect of supplementation of L-arginine (AR) on sub-fertile buffalo-bulls' ejaculates, 25 ejaculates of poor motility (40 to 55%) were collected by artificial vagina from 5 buffalo-bulls and extended with Tris-yolk extender (1:10) supplemented with different concentrations of AR (0, 3, 4, 5, and 6 mM). Semen was cooled gradually to 4 °C within 2 h and incubated at 4 °C for additional 2 h. Incubated semen samples were evaluated by computer-assisted semen analysis. Results showed that addition of 5 mM AR increased (P < 0.05) total sperm motility and rapid progressive motility percentages, while decreased (P < 0.05) non-motile sperm and static sperm percentages compared with AR-free (control) extender. Increasing the AR level to 6 mM increased (P < 0.05) the percentages of sperm progressive motility and rapid and slow progressive motilities, while decreased (P < 0.05) the non-progressive sperm motility percentages compared with AR-free extender. Supplementation of 5 mM AR improved (P < 0.05) sperm straight linear, curve linear, and average path velocities (36 ± 0.13, 20.6 ± 5.3, and 33.2 ± 8.5, respectively) in comparing with control and other AR treatments. Addition of AR (5 and 6 mM) improved (P < 0.05) the percentages of vitality (89.8 ± 1.9 and 80.0 ± 3.4, respectively), normality (44.3 ± 3.6 and 44.8 ± 1.5, respectively), and functional sperm (20.4 ± 8.6 and 21.0 ± 0.61, respectively), and decreased abnormal neck and tail percentages compared with AR-free extender. All AR levels decreased (P < 0.05) the abnormal neck and tail percentages. Addition of all AR levels had no significant (P > 0.05) effect on the activity of aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase in semen extender. Supplementation of Tris-yolk extender with L-arginine (5 or 6 mM) can improve sperm motility, velocity, vitality, and functional sperm and can decrease tail and neck abnormalities of sub-fertile buffalo ejaculate after 4 h incubation at cool temperature.


Assuntos
Arginina/farmacologia , Búfalos/fisiologia , Crioprotetores/farmacologia , Ração Animal/análise , Animais , Arginina/administração & dosagem , Arginina/metabolismo , Criopreservação/veterinária , Crioprotetores/administração & dosagem , Crioprotetores/metabolismo , Dieta/veterinária , Suplementos Nutricionais/análise , Relação Dose-Resposta a Droga , Masculino , Sêmen/efeitos dos fármacos , Análise do Sêmen/veterinária , Preservação do Sêmen/métodos , Preservação do Sêmen/veterinária , Motilidade Espermática/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos
20.
Trop Anim Health Prod ; 53(1): 61, 2021 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-33389175

RESUMO

This study was designed to evaluate the seasonal expression of seminal plasma proteins from two bovine breeds adapted to a subtropical climate and their associations with post-thawing sperm and environmental characteristics. Semen samples were obtained three times in summer and three times in winter from four Crioulo Lageano and four Angus bulls. Seminal plasma was obtained by centrifugation, and the other portion of the semen was cryopreserved. Seminal plasma proteins were identified by 2D-nanoUPLC-MSE. Post-thawing assessments of sperm kinetics, morphology and membrane integrity were performed. Environmental data such as air temperature, air humidity and black globe temperature (BGT) were recorded, and the temperature-humidity index (THI) was calculated in summer and winter. Results showed that the climate varied significantly between seasons. Although no statistical differences were observed in semen quality between breeds, the protein profiles varied within and between seasons. We suggest that the most critical proteins in summer affecting sperm characteristics were TIMP-2, DNase, Clusterin, CFAH and GPx6. TIMP-2 and DNase showed a higher abundance in Crioulo Lageano in comparison with Angus, while Clusterin, CFAH and GPx6 presented a lower abundance. To the best of our knowledge, this is the first report of a recently evolved type of glutathione peroxidase, GPx6, in seminal plasma of bovines. In winter, five proteins were considered to be more critical: BSP1, BSP3, CCL2, Sulfhydryl oxidase and TIMP-2. BSP1 and TIMP-2 showed a lower abundance while BSP3, CCL2 and Sulfhydryl oxidase presented a higher abundance in this season in Crioulo Lageano in comparison with Angus.


Assuntos
Aclimatação , Bovinos/metabolismo , Estações do Ano , Proteínas de Plasma Seminal/metabolismo , Adaptação Fisiológica , Animais , Cruzamento , Criopreservação/veterinária , Umidade , Masculino , Mapas de Interação de Proteínas , Sêmen , Análise do Sêmen/veterinária , Espermatozoides , Temperatura
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