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1.
Methods Mol Biol ; 2218: 99-115, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33606226

RESUMO

Cryopreservation of sperm cells is currently the most efficient tool for managing large and small collections of valuable genetic resources. Cryopreservation minimizes expenses for animal and facility maintenance such as personnel, water, power, and space. It extends the time offspring can be produced from individual organisms, reduces the need to maintain live populations, provides flexibility for planning future experiments and research projects, and can prevent catastrophic loss of irreplaceable research lines. In this chapter, we present the sperm collection, dilution, cryopreservation, thawing, and in vitro fertilization procedures used at the Zebrafish International Resource Center (ZIRC).


Assuntos
Criopreservação/métodos , Preservação do Sêmen/métodos , Espermatozoides/citologia , Animais , Crioprotetores/farmacologia , Fertilização In Vitro/métodos , Masculino , Motilidade Espermática/efeitos dos fármacos , Motilidade Espermática/fisiologia , Espermatozoides/efeitos dos fármacos , Peixe-Zebra/fisiologia
2.
Animal ; 15(1): 100001, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33515997

RESUMO

Contamination of semen with urine and asynchronous maturation of males and females are main obstacles in artificial reproduction of pikeperch Sander lucioperca. The objective of this study was to overcome these obstacles using optimization of a procedure for short-term storage of pikeperch semen at 4°C using two immobilizing media (IM): (a) IM1, 180mM NaCl, 2.68mM KCl, 1.36mM CaCl2⋅2H2O and 2.38mM NaHCO3, 343mOsm/kg; and (b) IM2, 200mM NaCl, 2.68mM KCl, 1.36mM CaCl2⋅2H2O and 2.38mM NaHCO3, 381mOsm/kg. Undiluted sperm was used as the control. At 6h poststorage, there were no substantial changes in spermatozoa motility and velocity at 30s postactivation in all groups. Over 48h of storage, the highest spermatozoa motility and velocity were obtained in sperm diluted in IM2 compared to the other groups. IM2 could maintain a significantly higher ATP content of diluted sperm than IM1 and undiluted treatment for 2days. Similarly, the highest values of eyeing and hatching rates were observed in sperm diluted in IM2 compared to sperm in the other studied groups. It can be concluded that the obtained result is a novel and applicable approach to maintain semen quality of pikeperch during short-term storage, suggesting IM2 as a promising medium for short-term storage. The present study also opens possibilities for ensuring a reliable source of semen as a convenient approach for increasing genetic diversity in hatcheries.


Assuntos
Preservação do Sêmen , Sêmen , Animais , Feminino , Masculino , Análise do Sêmen/veterinária , Preservação do Sêmen/veterinária , Motilidade Espermática , Espermatozoides
3.
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
4.
Trop Anim Health Prod ; 53(1): 86, 2021 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-33411090

RESUMO

This study determined the effects of antioxidant supplementation and storage time at cool temperatures on the characteristics of epididymal camel spermatozoa. Camel testes were collected at the abattoir after animal slaughtering and kept at 4 °C during transportation and until processing (max 6 h). Spermatozoa were retrieved and diluted with SHOTOR extender, split in aliquots, supplemented with the following antioxidants: 200 µm/mL vitamin E, 1.0 g/L vitamin C, 1 µg/mL selenium nanoparticles, 50 µg/mL zinc nanoparticles, 2 µg/mL sodium selenite, and 100 µg/mL zinc sulfate, and stored at 4 °C for 2, 48, 96, and 144 h. The storage time significantly affected (P < 0.05) the sperms' motility and livability, the sperms' membrane integrity, and the percentages of cytoplasmic droplets as well as the percentage of morphologically normal spermatozoa. Epididymal sperm characteristics (progressive motility, livability, membrane integrity, and abnormalities) were significantly improved (P < 0.05) when the spermatozoa were diluted with antioxidants as compared with the control group, and the best additives were identified as nano-selenium, sodium selenite, nano-zinc, and zinc sulfate. In conclusion, adding nano-sized minerals or inorganic trace elements and vitamins maintained the progressive motility, livability, and membrane integrity, and decreased abnormalities and cytoplasmic droplet percentages of epididymal camel spermatozoa stored at 4 °C up to 144 h.


Assuntos
Antioxidantes/administração & dosagem , Camelus/fisiologia , Preservação do Sêmen/veterinária , Espermatozoides/efeitos dos fármacos , Animais , Antioxidantes/farmacologia , Ácido Ascórbico/administração & dosagem , Ácido Ascórbico/farmacologia , Masculino , Nanopartículas Metálicas , Distribuição Aleatória , Selênio/administração & dosagem , Selênio/farmacologia , Vitamina E/administração & dosagem , Vitamina E/farmacologia , Zinco/administração & dosagem , Zinco/farmacologia
5.
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
6.
Methods Mol Biol ; 2180: 365-377, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32797421

RESUMO

In modern livestock breeding, cryopreserved semen is routinely used for artificial insemination. Sperm cryopreservation allows for long-term storage of insemination doses and secures reproduction at a desired time point. In order to cryopreserve semen, it needs to be carefully processed to preserve its vital functions after thawing. In this chapter, we describe the processes involved in cryopreservation of bull, stallion, and boar sperm. These include preparation of diluents, dilution of sperm in primary and freezing extender, slow cooling from room temperature to 5 °C, packaging of insemination doses in straws, freezing at a defined cooling rate in liquid nitrogen vapor, cryogenic storage, and thawing. Two-step dilution approaches, with commonly used diluents, are presented, namely, TRIS-egg yolk (TEY) extender for bull sperm, skim milk (INRA-82) extender for stallion sperm, and lactose-egg yolk (LEY) extender for boar sperm. Furthermore, simple methods are presented for cooling and freezing of sperm at defined cooling rates.


Assuntos
Criopreservação/veterinária , Crioprotetores/farmacologia , Preservação do Sêmen/veterinária , Sêmen/efeitos dos fármacos , Sêmen/fisiologia , Animais , Bovinos , Criopreservação/métodos , Cavalos , Masculino , Preservação do Sêmen/métodos , Suínos
7.
Methods Mol Biol ; 2180: 379-399, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32797422

RESUMO

Cryopreservation protocols for semen exist for bird species used in animal production, fancy and hobby species, and wild bird species. Freezing of bird oocytes or embryos is not possible. Cryopreservation of avian semen is used for preserving (genetic diversity of) endangered species or breeds. Freezing semen can also be used in the breeding industry for maintaining breeding lines, as a cost-effective alternative to holding live birds. Success and efficiency of cryopreservation of bird semen differs among species and breeds or selection lines. This chapter describes important variables of methods for collecting, diluting, cold storage, and freezing and thawing of bird semen, notably the medium composition, cryoprotectant used and its concentration, cooling rate, freezing method, and warming method. Media and methods are described for freezing semen using either glycerol or DMA as cryoprotectant, which both are known in chicken and a number of other bird species to render adequate post-thaw fertility rates.


Assuntos
Criopreservação/veterinária , Crioprotetores/farmacologia , Preservação do Sêmen/veterinária , Sêmen/efeitos dos fármacos , Sêmen/fisiologia , Animais , Aves , Criopreservação/métodos , Masculino , Preservação do Sêmen/métodos
8.
Methods Mol Biol ; 2180: 401-412, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32797423

RESUMO

Germplasm cryobanking of transgenic rodent models is a valuable tool for protecting important genotypes from genetic drift, genetic contamination, and loss of breeding colonies due to disease or catastrophic disasters to the housing facilities as well as avoiding stress associated with domestic and international live animal shipment. Furthermore, cryopreservation of germplasm enhances management efficiencies by saving animal room space, reducing workload for staff, reducing cost of maintaining live animals, reducing the number of animals used to maintain a breeding colony, and facilitating transportation of genetics by allowing distribution of frozen germplasm rather than live animals which also reduces the risk of transfer of pathogens between facilities. Thus, effective long-term preservation methods of mouse spermatozoa are critical for future reconstitution of scientifically important mouse strains used for biomedical research.


Assuntos
Bancos de Espécimes Biológicos , Criopreservação/métodos , Crioprotetores/farmacologia , Genoma , Preservação do Sêmen/métodos , Sêmen/efeitos dos fármacos , Sêmen/fisiologia , Animais , Pesquisa Biomédica , Masculino , Camundongos
9.
Methods Mol Biol ; 2180: 413-425, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32797424

RESUMO

Marine invertebrates represent the vast majority of marine biodiversity; they are extremely diverse playing a key role in marine ecosystems, thus playing an important role at the socioeconomic level. Some invertebrates such as sea urchins, ascidians, and horse-shoe crabs are very well-known model organisms for research and biocompound discovery. In this chapter we revisit the importance of cryopreservation for the conservation and rational use in research, fisheries management, or aquaculture and provide comprehensive protocols for the cryopreservation of sperm, embryos, and larvae.


Assuntos
Criopreservação/veterinária , Crioprotetores/farmacologia , Embrião não Mamífero/citologia , Larva/citologia , Preservação do Sêmen/veterinária , Espermatozoides/citologia , Animais , Organismos Aquáticos , Criopreservação/métodos , Embrião não Mamífero/efeitos dos fármacos , Invertebrados , Larva/efeitos dos fármacos , Masculino , Preservação do Sêmen/métodos , Espermatozoides/efeitos dos fármacos
10.
Methods Mol Biol ; 2180: 427-436, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32797425

RESUMO

Spermatozoa cryopreservation is used for the management of infertility and some other medical conditions. Routinely applied cryopreservation techniques depend on permeating cryoprotectants and relatively slow freezing rates. Cryoprotectant-free vitrification is an alternative and cost-effective method that is based on rapid cooling of spermatozoa by direct plunging into a cooling agent to prevent lethal intracellular ice crystallization and the detrimental effects of high salt concentrations. One of the problems with this technique is that full sterilization of commercially produced liquid nitrogen, which could be contaminated with different pathogens, is not possible. Here we use a benchtop device for the production of sterile liquid air with the same temperature as liquid nitrogen (-195.7 °C). This has been used to develop aseptic technology for cryoprotectant-free vitrification of human spermatozoa.


Assuntos
Criopreservação/métodos , Crioprotetores/farmacologia , Congelamento , Preservação do Sêmen/métodos , Espermatozoides/citologia , Vitrificação , Humanos , Masculino , Transição de Fase , Espermatozoides/efeitos dos fármacos
11.
Methods Mol Biol ; 2180: 721-730, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32797445

RESUMO

Long-term preservation of mammalian sperm at suprazero temperatures is desired to save storage and space costs, as well as to facilitate transport of preserved samples. This can be accomplished by the freeze-drying of sperm samples. Although freeze-drying results in immotile and membrane-compromised sperm, intracytoplasmic sperm injection (ICSI) can be used to introduce such an immotile sperm into an oocyte and thus start the fertilization process. So far, it has been shown that improved freeze-drying protocols preserve chromosomal integrity and oocyte-activating factor(s) in rodent and mammalian species at 4 °C for several years and at ambient temperature for up to 1 year depending on species, which permits shipping freeze-dried samples at ambient temperature. This chapter concisely reviews freeze-drying of mammalian sperm first and then presents a simple freeze-drying protocol.


Assuntos
Técnicas de Cultura de Células/métodos , Crioprotetores/química , Liofilização/métodos , Oócitos/citologia , Preservação do Sêmen/métodos , Injeções de Esperma Intracitoplásmicas , Espermatozoides/citologia , Animais , Proliferação de Células , Células Cultivadas , Cromossomos , Humanos , Masculino
12.
Anim Sci J ; 91(1): e13428, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32677083

RESUMO

Boar cryopreserved semen is scarcely used for artificial insemination due to its quality which is largely reduced by membrane lipid peroxidation. This present study was designed to improve the post-thawed boar semen quality by determining the optimal level of sericin supplementation (antioxidants) in semen extender. Five levels of sericin supplementation between 0% and 1% (w/v) were examined. Semen was frozen by the liquid nitrogen vapor method, thawed slowly at 5°C for 5 min, and used for the evaluation of sperm quality. The results indicated 0.5%-1% sericin supplementation was more effective on maintenance of sperm viability, acrosome integrity, and mitochondrial functions during freezing-thawing. Moreover, 0.75% sericin supplementation was most protective toward total sperm motility and sperm progressive motility. Additionally, 0.25%-0.75% sericin supplementation significantly suppressed increases in the index of lipid peroxidation. In conclusion, 0.75% sericin is recommended as an alternative component of the freezing extender to improve cryopreserved boar semen. However, further research using AI will be necessary to demonstrate that this indication can be applied to the production of offspring in the farms.


Assuntos
Antioxidantes , Bombyx/química , Criopreservação/métodos , Crioprotetores , Análise do Sêmen/veterinária , Preservação do Sêmen/métodos , Sericinas , Motilidade Espermática/efeitos dos fármacos , Acrossomo/efeitos dos fármacos , Animais , Antioxidantes/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Preservação do Sêmen/efeitos adversos , Sericinas/farmacologia , Suínos
13.
Rev Int Androl ; 18(3): 117-123, 2020.
Artigo em Espanhol | MEDLINE | ID: mdl-32660697

RESUMO

OBJECTIVE: The main objective of this revision is to summarize the current existing evidence of the potential adverse effects of SARS-CoV-2 on the male reproductive system and provide the recommendations of the Asociación Española de Andrología, Medicina Sexual y Reproductiva (ASESA) concerning the implications of COVID-19 infection in the management of male infertilty patients and testicular endocrine dysfunction. METHODS: A comprehensive systematic literature search of the databases of PubMed, Web of Science, Embase, Medline, Cochrane and MedRxiv, was carried out. RESULTS: The presence of orchitis as a potential complication of the infection by SARS-CoV-2 has not yet been confirmed. One study reported that 19% of males with COVID-19 infection had scrotal symptoms suggestive of viral orchitis which could not be confirmed. It is possible that the virus, rather than infecting the testes directly, may induce a secondary autoimmune response leading to autoimmune orchitis. COVID-19 has been associated with coagulation disorders and thus the orchitis could be the result of segmental vasculitis. Existing data concerning the presence of the virus in semen are contradictory. Only one study reported the presence of RNA in 15.8% of patients with COVID-19. However, the presence of nucleic acid or antigen in semen is not synonyms of viral replication capacity and infectivity. It has been reported an increase in serum levels of LH in males with COVID-19 and a significant reduction in the T/LH and FSH/LH ratios, consistent with subclinical hypogonadism. CONCLUSIONS: The findings of recent reports related to the potential effects of COVID-19 infection on the male reproductive system are based on poorly designed, small sample size studies that provide inconclusive, contradictory results. Since there still exists a theoretical possibility of testicular damage and male infertilty as a result of the infection by COVID-19, males of reproductive age should be evaluated for gonadal function and semen analysis. With regard to the sexual transmission of the virus, there is not sufficient evidence to recommend asymptomatic couples to abstein from having sex in order to protect themselves from being infected by the virus. Additional studies are needed to understand the long-term effects of SARS-CoV-2 on male reproductive function, including male fertility potential and endocrine testicular function.


Assuntos
Betacoronavirus , Infecções por Coronavirus/complicações , Pandemias , Pneumonia Viral/complicações , Saúde Reprodutiva , Saúde Sexual , Adulto , Betacoronavirus/isolamento & purificação , Betacoronavirus/patogenicidade , Betacoronavirus/fisiologia , Hormônio Foliculoestimulante/sangue , Humanos , Hipogonadismo/sangue , Hipogonadismo/etiologia , Imunoglobulina G/análise , Leucócitos , Hormônio Luteinizante/sangue , Masculino , Orquite/etiologia , Orquite/virologia , Próstata/virologia , RNA Viral/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sêmen/virologia , Preservação do Sêmen , Espanha , Testículo/imunologia , Testículo/patologia , Testículo/virologia , Testosterona/sangue , Vasculite/etiologia , Adulto Jovem
14.
PLoS One ; 15(6): e0234339, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32516324

RESUMO

Hypothermic storage of boar semen provides the possibility to omit antibiotics from semen extenders so long as sperm quality is maintained and bacterial growth prevented. The objective of this study was to determine an optimal cooling-rate frame for boar semen preserved at 5°C in an antibiotic-free extender. Semen from eight boars extended in AndroStar® Premium was cooled from 30°C to 5°C using seven different cooling rates, ranging initially from 0.01 to 0.36°C min-1 and reaching 5°C between 2 h and 24 h after dilution. Sperm motility, membrane integrity, membrane fluidity, mitochondrial membrane potential and the response to the capacitation stimulus bicarbonate remained at a high level for 144 h at 5°C when the semen was initially cooled in a cooling-rate frame ranging from 0.01 to 0.09°C min­1 in the temperature zone from 30 to 25°C, followed by 0.02 to 0.06°C min-1 to 10°C and 0.01 to 0.02°C min­1 to the final storage temperature. A cooling rate of 0.07°C min-1 in the temperature zone from 30 to 10°C led to a reduced response to bicarbonate (P < 0.01) and fast cooling to 5°C within 1 h with a cooling rate of 0.31°C min-1 resulted in lower values (P > 0.05) of all sperm parameters. In a further experiment, slow cooling with a holding time of 6 h at 22°C induced after 6 h storage a temporary increase in Escherichia coli of 0.5 × 103 to 2.4 × 103 CFU mL-1 in the sperm-free inoculated extender. Overall, the load of mesophilic bacteria in the stored semen was below 6 × 103 CFU mL-1, a level that is not regarded as critical for sperm quality. In conclusion, appropriate cooling protocols were established for the antibiotic-free storage of boar semen at 5°C, allowing the application of hypothermic preservation in research and in artificial insemination.


Assuntos
Criopreservação/métodos , Preservação do Sêmen/métodos , Manejo de Espécimes/métodos , Animais , Secreções Corporais/efeitos dos fármacos , Líquidos Corporais/efeitos dos fármacos , Crioprotetores/farmacologia , Masculino , Sêmen/efeitos dos fármacos , Sêmen/metabolismo , Motilidade Espermática/efeitos dos fármacos , Espermatozoides/fisiologia , Sus scrofa/metabolismo , Suínos , Temperatura
15.
Acta Vet Scand ; 62(1): 31, 2020 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-32552825

RESUMO

BACKGROUND: Cryopreservation of stallion spermatozoa tends to cause plasma membrane damage due to the low ratio of cholesterol to phospholipids. Gums have been suggested as an alternative cryoprotectant to glycerol for stallion spermatozoa. Therefore, the present experiment was designed to verify whether the effect of addition of cashew gum (CG), or nanoparticles (NP) containing CG, to the extender before cooling on sperm quality in stallion semen. Ejaculates from 6 stallions were extended and split between six treatment groups (control, a-tocopherol [TOC], CG1, CG0.5, NP1 and NP0.5), stored in cryotubes at 4 °C. RESULTS: Aliquots were analysed by computer-assisted sperm motility analysis on the day of collection, and after 24 h and 48 h of cold storage. After 48 h, the total motility with NP1 (78.53 + 6.31%) was similar to control 85.79 + 6.31% at 0 h. The same pattern was observed for progressive motility. Membrane integrity assessed by flow cytometer was similar between control, TOC and G1 at all storage times. The DNA fragmentation in the control group increased at all time points, whereas chromatin integrity was maintained after 24 h in TOC and NP0.5 compared to 0 h. There was no increase in the proportion of live spermatozoa producing hydrogen peroxide, but there was a tendency for an increased proportion of spermatozoa in the live superoxide category in CG1 after 24 h cooled storage. CONCLUSIONS: The addition of CG or CG-derived NP to extender for stallion semen was not harmful to the sperm cells.


Assuntos
Anacardium/química , Crioprotetores/farmacologia , Cavalos/fisiologia , Nanopartículas/química , Preservação do Sêmen/veterinária , Sêmen/fisiologia , Animais , Crioprotetores/química , Gengiva/química , Masculino , Preservação do Sêmen/instrumentação
16.
Arq. bras. med. vet. zootec. (Online) ; 72(3): 729-736, May-June, 2020. tab
Artigo em Inglês | LILACS, VETINDEX | ID: biblio-1128889

RESUMO

This study investigated in vitro the efficacy of four different extenders (TES-TRIS and TRIS with LDL low-density lipoprotein at concentrations of 10 or 5%) on the longevity of buffalo sperm in the refrigeration process at 5ºC. Sperm motility was assessed every 24 hours up to 72 hours of incubation using computer assisted sperm analysis and sperm membrane integrity was examined by the hypoosmotic test (HOST) at T1, T24, T48 and T72 hours. Eleven buffaloes (1 ejaculate per buffalo) of the Murrah breed were used, ranging in age from 4 to 5 years. Immediately after collection, each ejaculate was fractionated into 4 aliquots, and each aliquot was diluted in one of four diluents to obtain 50x106SPTZ/mL. The samples were packed in 0.5mL straws and refrigerated (-0.25°C/min) to 5°C and maintained at this temperature until evaluation. Prior to evaluation the samples were heated at 37°C for 30 seconds. The statistical package used for analysis was STATA 12.0 "Statistical Analysis Software" and means were compared by the Friedman test (P<0.05). The results of sperm kinetics and HOST indicate that the TRIS diluent with 10% LDL could be a promising alternative for semen refrigeration at 5ºC, to be used in conventional and fixed time artificial insemination.(AU)


Este estudo investigou in vitro a eficácia de quatro diferentes extensores (TES-TRIS e TRIS com lipoproteína de baixa densidade - LDL, nas concentrações de 10 ou 5%) sobre a longevidade espermática de búfalos no processo de refrigeração a 5ºC. A motilidade espermática foi avaliada a cada 24 horas até 72 horas de incubação, por sistema computadorizado "CASA", e a integridade de membrana espermática foi examinada pelo teste hiposmótico (HOST) em T1, T24, T48 e T72 horas. Foram utilizados 11 búfalos (um ejaculado por búfalo) da raça Murrah, com idade variando de quatro a cinco anos. Imediatamente após a coleta, cada ejaculado foi fracionado em quatro alíquotas, e cada alíquota foi diluída em um dos quatro diluidores para a obtenção de 50x106 SPTZ/mL. As amostras foram envasadas em palhetas de 0,5 mL, refrigeradas (-0,25oC/minuto) até 5oC e mantidas nessa temperatura até a avaliação. Previamente à avaliação, as amostras foram aquecidas a 37oC por 30 segundos. O pacote estatístico utilizado para as análises foi o STATA 12.0 "Statistical Analysis Software", e as médias foram comparadas pelo teste de Friedman (P<0,05). Os resultados de cinética e HOST até o tempo de 48 horas indicam que o diluidor TRIS com 10% LDL seria uma alternativa promissora para a refrigeração do sêmen a 5ºC, a ser utilizado na inseminação artificial e na inseminação artificial em tempo fixo.(AU)


Assuntos
Animais , Masculino , Preservação do Sêmen/veterinária , Motilidade Espermática , Búfalos , Lipoproteínas LDL , Técnicas In Vitro , Inseminação Artificial , Técnicas de Diluição do Indicador/veterinária
17.
Res Vet Sci ; 131: 153-158, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32387554

RESUMO

The maintenance of high vitality and motility of ram's spermatozoa during storage at low temperatures has a crucial role for successful fertilization. This study evaluates the effect of the natural antioxidant oregonin on ram semen quality, stored at 5 °C for 48 h. Еighteen ejaculates (three repetitions for 6 ejaculates) from three local breed rams, collecting by artificial vagina, with volume > 1 ml, concentration > 1 × 109/ml and mass motility >3.5 were used for chilling. Each ejaculate was separated in two equal parts, diluted with Tris-glucose-glycerol-egg yolk extender with no oregonin or supplemented with 100µÐœ oregonin until adjustment of the sperm concentration to 200 × 106 cells/ml and stored at 5 °C for 48 h. The semen quality assessment was based on the main kinematic (by CASA analysis), morphological parameters (by BrightVit kit staining) and mitochondrial status (by MitoView staining) of the spermatozoa on 0, 24 and 48 h of storage, and on in vivo fertility test. Oregonin did not impair the morphology and kept sustained motility of ram spermatozoa stored at 5 °C for 48 h. The curvilinear velocity indicated faster movement of the oregonin treated sperms that corresponded with high percent of spermatozoa with active mitochondria in these samples. The fertilizing capacity of spermatozoa was preserved and pregnancy rate in the experimental group was 80% versus 60% in control. In conclusion, this study provides a new data about positive effect of the natural antioxidant oregonin, supplemented to the extender, on chilled ram semen quality, including fertilizing ability.


Assuntos
Alnus/química , Antioxidantes/farmacologia , Diarileptanoides/farmacologia , Preservação do Sêmen/veterinária , Ovinos , Espermatozoides/efeitos dos fármacos , Animais , Antioxidantes/química , Temperatura Baixa , Diarileptanoides/química , Gema de Ovo , Feminino , Fertilização , Inseminação Artificial/veterinária , Masculino , Casca de Planta , Gravidez , Análise do Sêmen , Contagem de Espermatozoides , Motilidade Espermática/efeitos dos fármacos
18.
Poult Sci ; 99(5): 2766-2774, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32359614

RESUMO

Artificial insemination is used in almost 95% of turkey reproductive flocks and is becoming more important in chickens, particularly broiler breeders, as well as in assisted reproduction of wild birds kept in breeding centers. Diluted semen is recommended for artificial insemination. Pooled semen samples collected twice a week by dorso-abdominal massage from 2 chicken lines: laying-ISA Brown (ISA-B) and meat type-Hubbard Flex (H-F) were divided into 5 parts: neat semen and diluted in 1:2 ratio with 4 extenders: basic EK; EK + 1 µg/mL organic selenium and 8 µg/mL vitamin E; EK + 10 mg/mL of royal jelly; and EK + 0.25 g/mL of lyophilized bovine colostrum. Diluted semen samples were evaluated after 15 min and then 24 h storage at 4°C. Sperm concentration, motility, motility parameters (with Sperm Class Analyzer), and morphology were evaluated in the neat semen, whereas in diluted and stored samples, the last 3 traits were determined. In case of both lines, dilution did not affect (P > 0.05) the number of live normal cells (78.0-81.1% in ISA Brown and 73.8-68.7% in Hubbard Flex) in relation to neat semen; however, bovine colostrum addition increased (P < 0.05) the percentage of bulb head sperm (5.7 vs. 10.0% and 12.1 vs. 17.6%, for ISA and Hubbard, respectively) and decreased sperm motility (67.4 vs. 92.9% and 67.3 vs. 98.5% for ISA and Hubbard). The 24 h storage of neat semen and semen diluted with colostrum caused (P < 0.05) the unfavorable changes in all evaluated traits and both chicken lines, whereas semen dilution with remaining extenders decreased the percentage of live normal cells (by 18.8-23.4% ISA and by 20.9-25.5% Hubbard) but did not affect sperm motility (81.5-87.6% for ISA and 81.1-96.6% for Hubbard). Sperm motility and motility parameters depended both on the extender and chicken line.


Assuntos
Galinhas/fisiologia , Crioprotetores/farmacologia , Preservação do Sêmen/veterinária , Sêmen/fisiologia , Animais , Inseminação Artificial , Análise do Sêmen/veterinária , Preservação do Sêmen/métodos
19.
J Dairy Sci ; 103(7): 6698-6705, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32359996

RESUMO

Quantifying the relative population of sperm cells bearing the X or Y chromosome in a sexed-semen sample has historically been limited to methods that are either low throughput and sensitive to user subjectivity (e.g., fluorescence in situ hybridization), conterminous (using the same technology to generate and confirm the sex skew), or relatively insensitive (including quantitative PCR with a change detection threshold of 2×). Customers pay a premium for sexed semen and should have access to reliable sex skew data, generated by an accurate, precise test that is orthogonal to the method used to generate sexed semen. Droplet digital PCR (ddPCR) has the capacity to provide an accurate and precise sex skew quantitation by subdividing a pool of template DNA into nanoliter-scale droplets containing either 1 or 0 copies of template DNA. Then PCR amplification is conducted in the droplets, and the number of copies of the amplicon of interest can be counted as the number of fluorescence-positive droplets based on classic quantitative PCR fluorescent reporters. We have optimized and validated a multiplexed ddPCR assay that uses this copy counting method to quantify the sex skew (ratio of X or Y chromosomes) in frozen-thawed bovine sexed semen. The assay interrogates at least 1,000 cells per sample well, quantifying X and Y chromosome copy numbers along with an autosomal gene, GAPDH, used as an internal assay control to confirm total cells counted. The ddPCR sex skew assay achieved a 0.5-percentage-point variance for %X or %Y with a broad linear detection range, from 10 to 95% X, and provided reproducible skew values across a range of 9 to 27 ng of genomic DNA input. This approach overcomes some limitations of other sex skew assays by quantifying absolute X and Y chromosome copy numbers, thus providing a rigorous, independent assessment of sex-skewed semen.


Assuntos
Bovinos/genética , Análise do Sêmen/veterinária , Análise para Determinação do Sexo/veterinária , Espermatozoides/citologia , Animais , DNA , Feminino , Hibridização in Situ Fluorescente/veterinária , Masculino , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Preservação do Sêmen , Cromossomo X , Cromossomo Y
20.
Pesqui. vet. bras ; 40(4): 306-314, Apr. 2020. tab
Artigo em Inglês | LILACS, VETINDEX | ID: biblio-1135625

RESUMO

The objective of this study was to evaluate the sperm quality obtained of domestic cats by electroejaculation and recovery of the tail of the epididymis after cooling at -1°C and 4°C for 24 and 48 hours. Twenty-nine adult cats (2 to 6kg) were used. Sperm collection was performed by electroejaculation (EEJ), and after 48 hours, the cats were orchiectomized, and sperm sample was obtained from the vas deferens and epididymis tail (EPD). The samples were diluted in ACP-117® extender, and the sperm characteristics were evaluated at three different moments: when still fresh, 24 and 48 hours after cooling. The objective of this study was to evaluate the sperm quality obtained of domestic cats by electroejaculation and recovery of the tail of the epididymis after cooling at -1°C and 4°C for 24 and 48 hours. Twenty-nine adult cats (2 to 6kg) were used. Sperm collection was performed by electroejaculation (EEJ), and after 48 hours, the cats were orchiectomized, and sperm sample was obtained from the vas deferens and epididymis tail (EPD). The samples were diluted in ACP-117® extender, and the sperm characteristics were evaluated at three different moments: when still fresh, 24 and 48 hours after cooling. In order to compare the two refrigeration temperatures, the first stage was to analyze if there was a difference between the harvesting techniques. After this, two experiments were conducted: in the first, sperm sample from 14 cats were used and the cooling was performed at -1°C; and in the second, sample from 15 cats were used and the sperm were refrigerated at 4°C. Sperm kinetics were evaluated by computerized analysis (CASA) and concentration by Neubauer chamber, spermatic morphology was evaluated by modified Karras staining, and membrane integrity was evaluated by eosin nigrosine. The results obtained were analyzed in R software, version 3.2.5 using the Mann-Whitney test for variables with abnormal distributions, considering significance at the level of 5%. In ejaculate samples, higher values of total morphological defects were observed after 24 and 48 hours of refrigeration at 4°C (P<0.022) compared to refrigeration at -1°C, using Friedman test. To quantify the decrease in sperm quality, parameter reductions were calculated among time points (F-24h/F-48h/24h-48h). In EPD samples, a greater reduction in sperm quality was detected after 24 hours of refrigeration at 4°C, both in motility and sperm kinetics and in the movement and velocity indices, compared to refrigeration at -1°C. Based on the results, it can be concluded that cooling of feline spermatozoa at -1°C for up to 48 hours was efficient in maintaining spermatic quality collected by EEJ and EPD, and it could be an alternative to spermatozoa cryopreservation in domestic felines.(AU)


O objetivo deste trabalho foi avaliar a qualidade espermática de gatos domésticos obtidos por eletroejaculação e recuperação da cauda do epidídimo após a refrigeração a -1°C e a 4°C por 24 e 48 horas. Vinte e nove gatos adultos (2 a 6kg) foram utilizados. A colheita de espermatozoides foi realizada por eletroejaculação (EEJ) e, após 48 horas, os gatos foram orquiectomizados, e as amostras espermáticas foram obtidas a partir do ducto deferente e da cauda do epidídimo (EPD). As amostras foram diluídas em ACP-117® e as características espermáticas foram avaliadas em três momentos distintos: fresco, 24 e 48 horas após a refrigeração. Para ser possível comparar as duas temperaturas de refrigeração, a primeira etapa foi analisar se havia diferença entre as técnicas de colheita. Após isto, dois experimentos foram conduzidos: no primeiro, espermatozoides de 14 gatos foram utilizados e a refrigeração foi realizada a -1°C; e no segundo, amostras de 15 gatos foram utilizados e os espermatozoides foram refrigerados a 4°C. A cinética espermática foi avaliada por análise computadorizada (CASA), a concentração por câmara de Neubauer, a morfologia espermática foi avaliada pela coloração de Karras modificada, e a integridade da membrana foi avaliada por eosina nigrosina. Os resultados obtidos foram analisados no software R, versão 3.2.5, utilizando o teste de Mann-Whitney para variáveis com distribuições anormais, considerando significância ao nível de 5%. No ejaculado, maiores valores de defeitos morfológicos totais foram observados após 24 e 48 horas de refrigeração a 4°C (P<0,022) em comparação com refrigeração a -1°C, usando o teste de Friedman. Para quantificar a diminuição na qualidade espermática, as reduções dos parâmetros foram calculadas entre os pontos de tempo (F-24h/F-48h/24h-48h). Na EPD, uma maior redução na qualidade espermática foi detectada após 24 horas de refrigeração a 4°C, tanto na motilidade e na cinética espermática quanto nos índices de movimento e velocidade, em comparação com a refrigeração a -1°C. Com base nos resultados, pode concluir-se que a refrigeração dos espermatozoides felino a -1°C, até 48 horas, foi eficaz na manutenção da qualidade espermático colhidos por EEJ e EPD, e pode ser uma alternativa para a criopreservação de espermatozoides em felinos domésticos.(AU)


Assuntos
Animais , Masculino , Gatos , Sêmen , Preservação do Sêmen/veterinária , Espermatozoides , Criopreservação , Técnicas de Reprodução Assistida , Técnicas de Reprodução Assistida/veterinária , Epididimo
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