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1.
Theriogenology ; 223: 29-35, 2024 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-38663138

RESUMO

The freeze-thawing process induces osmotic changes that may affect the membrane domain location of aquaporins' (AQP) in spermatozoa. Recent studies suggest that changes in AQP3 localization allows better sperm osmo-adaptation, improving the cryoresistance. Ultra-rapid freezing is an alternative cryopreservation technique that requires less equipment than conventional freezing, and it is faster, simpler and can be used in the field. This study aimed to determine the influence of freezing-thawing rates (slow (control) vs. ultra-rapid) on AQP3 expression and location in the spermatozoa from small ruminants (sheep and goats) and its relationship with sperm cryo-damage. Spermatozoa were collected from 10 Merino rams and 10 Murciano-Granadina bucks. The presence and distribution of AQP3 were assessed by Western blotting and immunocytochemistry (ICC), employing a commercial rabbit polyclonal antibody. Sperm motility was CASA system-analyzed, and membrane and acrosome integrity assessed by fluorescence (PI/PNA-FITC). Western blotting did not detect a significant effect of freezing-thawing rate on the amount of AQP3 while ICC found freezing-thawing rate affecting AQP3 location (P < 0.05). In both species, the percentages of spermatozoa showing AQP3 in the post-acrosome region, mid-piece, and principal piece of the tail were greater in samples cryopreserved by slow freezing-thawing (control) than ultra-rapid freezing-thawing rates (P < 0.05). Spermatozoa cryopreserved using ultra-rapid freezing-thawing showed decrease motility, plasma membrane, and acrosome integrity (P < 0.05), which might be related, at least in part, to a lower expression of AQP3. In conclusion, the cooling rate modifies the location of AQP3 in spermatozoa of sheep and goat, which might be associated with sperm cryosurvival.


Assuntos
Aquaporina 3 , Criopreservação , Cabras , Preservação do Sêmen , Espermatozoides , Animais , Masculino , Cabras/fisiologia , Aquaporina 3/metabolismo , Espermatozoides/fisiologia , Espermatozoides/metabolismo , Criopreservação/veterinária , Ovinos/fisiologia , Preservação do Sêmen/veterinária , Preservação do Sêmen/métodos , Congelamento , Motilidade dos Espermatozoides
2.
Biopreserv Biobank ; 21(2): 142-148, 2023 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35675676

RESUMO

Aim: This work examined the influence of induced changes in prolactin (PRL) secretion on sperm cryoresistance of ibex and the mouflon. Materials and Methods: PRL secretion was modified in a first experiment by the use of bromocriptine (BCR, dopamine agonist) during the non-breeding season, and in a second experiment by the use of sulpiride (SLP, dopamine D2-receptor antagonist) during the rutting season. Slow and ultra-rapid freezing protocols were used to cryopreserve sperm samples. Results: BCR decreased blood plasma PRL concentrations, whereas SLP increased them. Cryoresistance ratios (CRs) for curvilinear velocity (VCL), straight-line velocity (VSL), and average path velocity (VAP) in BCR-treated mouflons were lower than in controls using slow-freezing (p < 0.05), while CRs of motility and morphologically normal sperm of BCR-treated mouflons were greater than controls with ultra-rapid freezing (p < 0.05). BCR increased the head sperm dimensions in ibexes (p < 0.001); conversely, BCR decreased the head dimensions in mouflons (p < 0.001). CR-motility, CR-amplitude of lateral head displacement (ALH), CR-viability, and CR-acrosome integrity in SLP-treated mouflons were lower than in controls with slow-freezing (p < 0.01); CR-viability and CR-acrosome were lower than controls with ultra-rapid freezing (p < 0.05). In ibexes, CR-ALH was lower for SLP-treated (p < 0.05). SLP treatment increased head dimensions in ibexes (p < 0.001) but did not affect the sperm head of mouflons. Conclusion: Our findings show that high levels of blood plasma PRL negatively affect the cryoresistance of ibex and mouflon sperm.


Assuntos
Preservação do Sêmen , Carneiro Doméstico , Animais , Masculino , Prolactina , Sêmen , Criopreservação/métodos , Motilidade dos Espermatozoides , Preservação do Sêmen/métodos , Espermatozoides , Acrossomo , Cabras
3.
Vet Med Sci ; 8(3): 1311-1318, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35419978

RESUMO

BACKGROUND: The Cantabrian capercaillie (Tetrao urogallus cantabricus) is critically endangered. This subspecies has the lowest genetic variability and it is in regression. It belongs to Phasianidae family; therefore, the domestic chicken (Gallus gallus domesticus) could be a good model for developing reproductive technologies for use in capercaillie populations with low availability of animals. OBJECTIVES: In this study, we analyzed the response of capercaillie sperm to the freezing-thawing process for contributing to the development of a semen cryobank of Cantabrian capercaillie. METHODS: We used domestic chicken as the animal model in order to obtain the freezing protocol before applying on capercaillie. In the first experiment, two different extenders (EK and LR84) and different concentrations [4% and 6% dimethyl-acetamide (DMA) v:v] of cryoprotectants were evaluated using in-straw freezing method in domestic chickens. A pilot study in capercaillie males, using the same conditions evaluated in chicken, was performed. RESULTS: In chicken, we found that the LR84-4% DMA media provided the best results for freezing semen. In capercaillie study, LR84 extender seemed to be the most appropriate diluent and 4% was the better dose of DMA cryoprotectant agent. Further, based on previous studies carried out in rooster samples, we also tested the glycerol (8% v/v) as a cryoprotectant for capercaillie semen cryopreservation. CONCLUSIONS: Our results suggest that sperm from both domestic and wild species had a similar response to freezing-thawing processes. Mediterranean chickens may be used as a suitable model for developing sperm freezing protocols that can be extrapolated to threatened capercaillie populations. In addition, LR84 media with glycerol was the most efficient extender to freeze capercaillie sperm native.


Assuntos
Galinhas , Preservação do Sêmen , Animais , Galinhas/fisiologia , Criopreservação/métodos , Criopreservação/veterinária , Crioprotetores/farmacologia , Glicerol , Masculino , Projetos Piloto , Melhoramento Vegetal , Sementes , Análise do Sêmen/veterinária , Preservação do Sêmen/métodos , Preservação do Sêmen/veterinária , Motilidade dos Espermatozoides
4.
Front Vet Sci ; 9: 1114695, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36727036

RESUMO

Introduction and objective: Cryopreservation of testicular tissues offers new possibilities to protect endangered species, genetically valuable individuals or even the fertility potential of prepubertal individuals who have died unexpectedly. However, the use of this technique still remains a challenge. In this study, slow freezing and vitrification of testicular tissue was investigated to find out which cryopreservation method could better preserve the viability and DNA integrity of testicular germ cells in diverse wild species. Methods: Testes were obtained post-mortem from 18 artiodactyls (wild boar, roe deer, dwarf goat, mhor gazelle, European mouflon, African forest buffalo, Malayan tapir, dorcas gazelle, Iberian ibex, gnu, red river hog), 5 primates (colobus monkey, capuchin monkey, mandrill), 8 carnivores (gray wolf, Persian leopard, binturong, European mink, American black bear, suricata), and 2 rodents (Patagonian mara). The testicles belonged to adult individuals and were cut into small pieces and cryopreserved by needle immersed vitrification or uncontrolled slow freezing using a passive cooling device. After warming or thawing, testicular tissues were enzymatically digested and two germ cell types were differentiated based on their morphology: rounded cells (spermatogonia, spermatocytes, and early spermatids) and elongated cells (elongated spermatids and spermatozoa). Cell viability was assessed by SYBR-14/propidium iodide while DNA fragmentation by TUNEL assay with fluorescence microscope. Results and discussion: Our preliminary results revealed that our uncontrolled slow freezing method better preserved the viability and DNA integrity of elongated cells than vitrification. Such trend was observed in all species, being significant in artiodactyls, carnivores, and primates. Similarly, the viability and DNA integrity of rounded cells was also better maintained in primates by uncontrolled slow freezing, while in carnivores, vitrification by needle immersion showed better results in this type of cells. In artiodactyls and rodents both techniques preserved the viability of rounded cells in a similar manner, although the DNA integrity of these cells was greater after needle immersed vitrification in artiodactyls. Conclusions: In conclusion, the effectiveness of each cryopreservation method is affected by the phylogenetic diversity between species and cell type.

5.
Andrology ; 9(4): 1242-1253, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33686789

RESUMO

BACKGROUND: Recent studies have noted that the circulating testosterone concentration may affect the ability of spermatozoa to survive cryopreservation. However, few attempts to confirm such a relationship have been made. Wild ruminant species have very marked seasonal changes in their reproductive function and strong annual changes in their plasma testosterone concentration. OBJECTIVES: The present work examines the influence of induced changes in testosterone secretion on sperm variables following conventional slow freezing and ultra-rapid freezing, using the Iberian ibex as an experimental model. MATERIALS AND METHODS: In a first experiment, testosterone levels were reduced in the middle of the rutting season (December) using the antiandrogen cyproterone acetate (CA). In a second experiment, testosterone levels were increased at the end of the rutting season (January) via the use of the androgen testosterone propionate (TP). RESULTS: During December, the testosterone concentration was found to be higher in the blood and seminal plasma of untreated males than in those of CA-treated males (p < 0.001 and p < 0.05, respectively). Compared with controls, the TP-treated animals had higher blood plasma testosterone concentrations but lower seminal plasma testosterone concentrations during January (p < 0.01 and p < 0.001, respectively). The seminal vesicles of the TP-treated males were larger than those of untreated males (p < 0.05). When CA was administered, sperm viability improved compared with controls (p < 0.05), irrespective of the freezing protocol followed. For the ultra-rapid freezing procedure, the cryoresistance ratio for motility decreased when TP was administered (p < 0.05). The values for fresh sperm morphometric variables decreased during the 50 days after the end of CA treatment (p < 0.001) and increased over the same time after the end of TP treatment (p < 0.001). DISCUSSION AND CONCLUSION: The circulating testosterone concentration appears to influence sperm cryoresistance. This may explain the seasonal changes seen in sperm freezability in some species, independent of fresh sperm quality.


Assuntos
Criopreservação/métodos , Cabras , Modelos Teóricos , Espermatozoides , Testosterona/sangue , Animais , Masculino , Preservação do Sêmen/métodos
6.
Animals (Basel) ; 11(1)2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33467758

RESUMO

The American flamingo is a useful model for the development of successful semen cryopreservation procedures to be applied to threatened related species from the family Phoenicopteridae, and to permit genetic material banking. Current study sought to develop effective sperm cryopreservation protocols through examining the influences of two permeating cryoprotectants and the seminal plasma removal. During two consecutive years (April), semen samples were collected and frozen from American flamingos. In the first year, the effect of two permeating cryoprotectants, DMA (dimethylacetamide) (6%) or Me2SO (dimethylsulphoxide) (8%), on frozen-thawed sperm variables were compared in 21 males. No differences were seen between DMA and Me2SO for sperm motility, sperm viability, and DNA fragmentation after thawing. In the second year, the role of seminal plasma on sperm cryoresistance was investigated in 31 flamingos. Sperm samples were cryopreserved with and without seminal plasma, using Me2SO (8%) as a cryoprotectant. The results showed that samples with seminal plasma had higher values than samples without seminal plasma for the following sperm variables: Straight line velocity (22.40 µm/s vs. 16.64 µm/s), wobble (75.83% vs. 69.40%), (p < 0.05), linearity (62.73% vs. 52.01%) and straightness (82.38% vs. 73.79%) (p < 0.01); but acrosome integrity was lower (55.56% vs. 66.88%) (p < 0.05). The cryoresistance ratio (CR) was greater in samples frozen with seminal plasma than without seminal plasma for CR-progressive motility (138.72 vs. 54.59), CR-curvilinear velocity (105.98 vs. 89.32), CR-straight line velocity (152.77 vs. 112.58), CR-average path velocity (122.48 vs. 98.12), CR-wobble (111.75 vs. 102.04) (p < 0.05), CR-linearity (139.41 vs. 113.18), and CR-straightness (124.02 vs. 109.97) (p < 0.01). This research demonstrated that there were not differences between Me2SO and DMA to successful freezing sperm of flamingos; seminal plasma removal did not provide a benefit for sperm cryopreservation.

7.
Vet Med Sci ; 7(1): 256-263, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33040487

RESUMO

The domestic ferret (Mustela putorius furo) provides a good model for developing new reproductive technologies for use with threatened related species. Such technologies could also be used in the reproductive management of this pet species. The present work reports an improved freezing protocol for ferret sperm. Semen was collected by electroejaculation plus rectal massage (in an attempt to reduce the electrical stimulation necessary) from five adult male ferrets, and then subjected to one of two freezing protocols: (a) from 5 to -35°C at 40°C/min, then from -35 to -65°C at 17°C/min, and finally from -65 to -85°C at 3°C/min-a decelerating freezing rate; and (b) from 5 to - 10°C at 5°C/min, and then from -10 to -130°C at 60°C/min-an accelerating freezing rate. After thawing, the viability and acrosomal integrity of the sperm frozen via the two-step accelerating method were better than those frozen via the three-step decelerating method (43.3 ± 3.5% and 71.2 ± 3.4% compared with 29.7 ± 3.7% and 58.8 ± 3.4% respectively; p < .05). No differences were seen between the methods with respect to sperm motility variables; most sperm (>90%) remained static with both freezing methods. In conclusion, although the method with accelerating freezing rate was associated with better post-thaw sperm viability and acrosome integrity values, neither of the two freezing methods tested provided adequate motility results after thawing. Combining rectal massage with electrical stimuli seemed to reduce the number of the latter required for successful sperm collection.


Assuntos
Criopreservação/veterinária , Congelamento , Preservação do Sêmen/veterinária , Animais , Criopreservação/instrumentação , Criopreservação/métodos , Ejaculação/fisiologia , Furões/fisiologia , Massagem/veterinária , Preservação do Sêmen/instrumentação , Preservação do Sêmen/métodos
8.
Animals (Basel) ; 10(4)2020 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-32316152

RESUMO

Sperm cryopreservation is a complex process that needs to be adapted to wild and domestic avian species to ensure proper efficiency. Because of its accessibility, the peregrine falcon may be used as a good model for studying other raptor species. To find the most optimal cryopreservation protocol for peregrine falcon ejaculates, sperm parameters such as motility, viability, DNA fragmentation, acrosome integrity, and mitochondrial activity were analyzed under different conditions by varying the freezing method (slow freezing in straws vs. ultrarapid freezing in pellets), thawing conditions (37 °C for 30 s vs. 5 °C for 1 min), type of cryoprotectant (DMA vs. DMSO), and concentration of DMSO (4% vs. 8%). Results show that slow cryopreservation in straws yielded greater percentages (p < 0.05) of motile spermatozoa (22.5% ± 4.4% vs. 0.0% ± 4.1%), viable spermatozoa with intact acrosomes (84.6% ± 4.3% vs. 77.4% ± 4.3%), and spermatozoa with active mitochondria (41.0% ± 6.7% vs.12.8% ± 6.7%), compared with those obtained by the ultrarapid freezing in pellets. However, no differences were found between different thawing conditions. Moreover, all sperm motility parameters were greater (p < 0.05) when DMSO was used during freezing compared with DMA, although the use of 3% and 8% DMSO produced similar results. In conclusion, these results represent important progress in the study of falcon semen cryopreservation protocol, highlighting the crucial steps of the process and the most suitable conditions.

9.
J Zoo Wildl Med ; 51(1): 196-201, 2020 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-32212563

RESUMO

This article describes the urinogenital condition of three female Iberian ibexes (Capra pyrenaica-one infertile 3-yr-old adult and two prepubertal animals aged 1 (PP1) and 2 (PP2) yr, respectively, all raised in captivity. All showed constant urinal dribbling, leading to ulcerative dermatitis in the vulvar area. Housed in a stable with other females, the adult did not become pregnant after male contact in either of two consecutive mating seasons. Vaginoscopy and laparoscopic exploration performed on the prepubertal females revealed abnormalities of the vagina and urinary bladder. Ultrasound examination revealed atrophy of the left kidney in the adult female and PP1, and of the right kidney in PP2, with degeneration of the renal pelvis. A paraovarian cyst with hydrosalpinx was also detected in the left oviduct of the adult female. Postmortem analysis of the adult and PP2, which shared a mother, confirmed an extramural single ectopic ureter with vaginal insertion associated with atrophy of the ipsilateral kidney. Though PP1 was officially unrelated to the latter animals, all three might have had a common ancestor in their lineages.


Assuntos
Infertilidade/veterinária , Nefropatias/veterinária , Ureter/anormalidades , Animais , Animais de Zoológico , Atrofia/patologia , Atrofia/veterinária , Feminino , Cabras/anormalidades , Infertilidade/etiologia , Nefropatias/diagnóstico , Nefropatias/patologia , Espanha , Ureter/patologia
10.
PLoS One ; 15(1): e0227946, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31978160

RESUMO

Sperm cryopreservation by ultra-rapid cooling based on dropping small volumes of sperm suspension directly into liquid nitrogen, has been successful in some wild ruminant species, including the Iberian ibex (Capra pyrenaica). In ultra-rapid cooling, the contents of these droplets are expected to enter a stable, glass-like state, but to the best of our knowledge no information exists regarding the presence or absence of ice formation in the extracellular milieu when using this technique. Different modifications to the extracellular milieu likely inflict different types of damage on the plasmalemma, the acrosome and mitochondrial membranes. The aims of the present work were: 1) to examine the physical state of the extracellular milieu after cryopreservation at slow and ultra-rapid cooling rates-and thus determine whether ultra-rapid cooling vitrifies the extracellular milieu; and 2) to compare, using conventional sperm analysis techniques and scanning and transmission electron microscopy, the damage to sperm caused by these two methods. Sperm samples were obtained by the transrectal ultrasound-guided massage method (TUMASG) from anesthetized Iberian ibexes, and cryopreserved using slow and ultra-rapid cooling techniques. Sperm motility (22.95 ± 3.22% vs 4.42 ± 0.86%), viability (25.64 ± 3.71% vs 12.8 ± 2.50%), acrosome integrity (41.45± 3.73% vs 27.00 ± 1.84%) and mitochondrial membrane integrity (16.52 ± 3.75% vs 4.00 ± 0.65%) were better after slow cooling (P<0.001) than after ultra-rapid technique. Cryo-scanning electron microscopy (Cryo-SEM) suggested that the vitrified state was not achieved by ultra-rapid cooling, and that the ice crystals formed were smaller and had more stretchmarks (P<0.001) than after slow cooling. Scanning electron microscopy revealed no differences in the types of damage caused by the examined techniques, although transmission electron microscopy showed the damage to the plasmalemma and mitochondrial membrane to be worse after ultra-rapid cooling. In conclusion ultra-rapid cooling provoked more membrane damage than slow cooling, perhaps due to the extracellular ice crystals formed.


Assuntos
Cabras/genética , Análise do Sêmen/métodos , Preservação do Sêmen/métodos , Espermatozoides/fisiologia , Acrossomo/efeitos dos fármacos , Acrossomo/ultraestrutura , Animais , Temperatura Baixa , Criopreservação , Crioprotetores/farmacologia , Humanos , Masculino , Motilidade dos Espermatozoides/efeitos dos fármacos , Vitrificação/efeitos dos fármacos
11.
PLoS One ; 14(1): e0209910, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30608977

RESUMO

Seminal plasma is a key biological fluid that modulates sperm function in the reproduction process. However, its role in sperm biotechnologies is scarce in poultry. The aims of the present study were to study the amino acids profile and total proteins of seminal plasma in 12 Spanish chicken breeds and to investigate the role of seminal plasma on cryoresistance of rooster sperm. To investigate the role of seminal plasma on cryoresistance, diluted pooled semen samples were cryopreserved in the presence and absence of seminal plasma. Glutamic acid was the most abundant free amino acid in seminal plasma, followed by alanine, serine, valine, and glycine. There was an influence of breed (P<0.05) on the percentage of viable sperm after freezing-thawing of samples with seminal plasma. Cluster analysis revealed that White Prat, Black Castellana, Blue Andaluza, Quail Castellana, and Red-Barred Vasca returned the best freezing-thawing response (good freezers). There was a positive correlation between seminal plasma concentrations of valine, isoleucine lysine, leucine and post thaw viability. The evaluation of fertilization capacity of frozen-thawed semen from the breeds White Prat ('good freezer') and Black-Red Andaluza ('bad freezer') showed that good freezer had higher fertility (20/68, 29.4%) compared to bad freezer breed (14/76, 18.4%), even if the difference was not significant (P = 0.08). The TUNEL assay revealed that freezing/thawing procedures in presence of seminal plasma provoked higher DNA fragmentation in most of the breeds, with a positive correlation between seminal alanine, valine, isoleucine, methionine, leucine, tyrosine, phenylalanine concentrations and DNA integrity. DNA fragmentation was lower in absence of seminal plasma and the breed effect on sperm viability was highly reduced. It is concluded that specific seminal plasma amino acids were associated with post-thaw percentage of viable sperm and DNA integrity. The removal of seminal plasma decreases the variability of the results and DNA fragmentation damages.


Assuntos
Aminoácidos/sangue , Sêmen/fisiologia , Espermatozoides/fisiologia , Alanina/sangue , Animais , Galinhas , Criopreservação/métodos , Fragmentação do DNA , Ácido Glutâmico/sangue , Glicina/sangue , Marcação In Situ das Extremidades Cortadas , Masculino , Serina/sangue , Valina/sangue
12.
Cryobiology ; 80: 12-17, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29352980

RESUMO

The rate at which lethal intracellular ice forms during sperm cryopreservation is highly dependent on the cooling protocol. The present work compares two cooling protocols for use with Iberian ibex (Capra pyrenaica) sperm by assessing the effects on the motility, viability, and size of frozen-thawed sperm cells. Ejaculates, obtained from six adult ibex males via transrectal, ultrasound-guided massage of the accessory sex glands plus electroejaculation if necessary, were cooled via either 1) Protocol 1 (decelerating cooling), involving cooling in liquid nitrogen vapor from 5 °C to -35 °C (40 °C/min), from -35 °C to -65 °C (17 °C/min), and then from -65 °C to -85 °C (3 °C/min); or 2) Protocol 2 (accelerating cooling) involving cooling in a biological freezer from 5 °C to -5 °C (4 °C/min), from -5 °C to -110 °C (25 °C/min), and then from -110 °C to -140 °C (35 °C/min). Compared to fresh ejaculates, sperm quality at thawing was found to be reduced by both protocols (p < .05), but especially by Protocol 1. Sperm head size was also significantly reduced by both protocols, although the Protocol 1 sperm heads were also significantly smaller than those of Protocol 2 sperms heads (p < .05). In fresh sperm samples, clustering analyses revealed two subpopulations of sperms with different morphometric characteristics, SP1 with larger cells, and SP2 with smaller cells. Both cooling protocols caused reduction in the proportion of SP1 cells, and an increase in the proportion of SP2 cells. In conclusion, the decelerating cooling protocol (Protocol 1) caused greater cryodamage to the sperm cells than the accelerating protocol (Protocol 2).


Assuntos
Criopreservação/métodos , Preservação do Sêmen/métodos , Espermatozoides , Animais , Criopreservação/veterinária , Cabras , Masculino , Preservação do Sêmen/veterinária , Cabeça do Espermatozoide , Temperatura
13.
Asian J Androl ; 18(6): 882-888, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27678467

RESUMO

Postcopulatory sexual selection through sperm competition may be an important evolutionary force affecting many reproductive traits, including sperm morphometrics. Environmental factors such as pollutants, pesticides, and climate change may affect different sperm traits, and thus reproduction, in sensitive bird species. Many sperm-handling processes used in assisted reproductive techniques may also affect the size of sperm cells. The accurately measured dimensions of sperm cell structures (especially the head) can thus be used as indicators of environmental influences, in improving our understanding of reproductive and evolutionary strategies, and for optimizing assisted reproductive techniques (e.g., sperm cryopreservation) for use with birds. Computer-assisted sperm morphometry analysis (CASA-Morph) provides an accurate and reliable method for assessing sperm morphometry, reducing the problem of subjectivity associated with human visual assessment. Computerized systems have been standardized for use with semen from different mammalian species. Avian spermatozoa, however, are filiform, limiting their analysis with such systems, which were developed to examine the approximately spherical heads of mammalian sperm cells. To help overcome this, the standardization of staining techniques to be used in computer-assessed light microscopical methods is a priority. The present review discusses these points and describes the sperm morphometric characteristics of several wild and domestic bird species.


Assuntos
Técnicas de Reprodução Assistida , Espermatozoides/citologia , Animais , Aves , Forma Celular/fisiologia , Masculino , Análise do Sêmen/métodos , Preservação do Sêmen/métodos
14.
Theriogenology ; 84(7): 1067-74, 2015 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-26174035

RESUMO

The objective was to characterize the stress response and the seminal parameters obtained with electroejaculation (EE) or transrectal ultrasound-guided massage of the accessory sex glands (TUMASG) in two captive but nondomestic ruminants, the mouflons and the Iberian ibex under general anesthesia. In mouflons, the physiological responses (heart and respiratory rate, rectal temperature, cortisol, creatine kinase, potassium and glucose concentrations) changed similarly with both procedures. The TUMASG procedure was faster than EE in mouflons (21.7 ± 1.4 vs. 12.4 ± 1.2 minutes, P < 0.01). In ibexes, respiratory rate, cortisol and creatine kinase concentration changes were greater with EE than with TUMASG (final respiratory rate: 62.7 ± 5.5 vs. 38.1 ± 5.6 breaths/min [P < 0.05]; final cortisol: 51.4 ± 5.1 vs. 25.3 ± 5.6 ng/mL [P < 0.001]; and final creatine kinase: 300.9 ± 99.9 vs. 87.1 ± 16.9 U/L [P < 0.001]). Electroejaculation provided better results in some sperm parameters (mouflons: sperm score: 3.4 ± 0.3 vs. 2.6 ± 0.2 [P < 0.01]; total number of sperm ejaculated: 982.4 ± 299 vs. 710.0 ± 542.2 [P < 0.05]; ibexes: sperm with progressive motility: 47.7 ± 6.2 vs. 20.5 ± 8.3 [P < 0.05]). The transrectal ultrasound-guided massage of the accessory sex glands appears to be an alternative technique to collect sperm from wild ruminants, reducing the need for electrical stimuli and thus decreasing the undesired responses of EE in the more sensitive species. On the other hand, better fresh sperm may be collected with EE. However, TUMASG provides practical advantages in animal welfare, firstly in these wild species more sensible to stress management and capture myopathy.


Assuntos
Cabras/fisiologia , Carneiro Doméstico/fisiologia , Manejo de Espécimes/veterinária , Espermatozoides/fisiologia , Animais , Glicemia/análise , Creatina Quinase/sangue , Ejaculação , Estimulação Elétrica , Genitália Masculina , Frequência Cardíaca , Hidrocortisona/sangue , Masculino , Massagem , Potássio/sangue , Reto , Taxa Respiratória , Manejo de Espécimes/métodos , Contagem de Espermatozoides/veterinária , Motilidade dos Espermatozoides
15.
J Zoo Wildl Med ; 46(2): 335-8, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26056889

RESUMO

This paper describes the sperm characteristics and response to cooling and freezing of naturally ejaculated semen from a captive, adult golden eagle (Aquila chrysaetus) trained to allow sperm recovery via cooperative copulation. A basic spermiogram was prepared, and sperm motility and morphometric variables recorded using a computer-aided system. For sperm storage, the effects of a polyvinylpyrrolidone-based extender were evaluated at 5°C. The same extender was also used in freezing procedures in which glycerol (11%) and dimethylacetamide (6%) were compared as cryoprotectants. The extender preserved sperm viability over storage periods of up to 6 days. Although sperm motility and percentage live sperm values were poorer for frozen-thawed (5.8-14.6% and 44-42%, respectively) than for fresh samples (46.7 and 74.6%, respectively), no differences were seen between the effects of the two cryoprotectants. These results could be of use when attempting to store the sperm of golden eagles and other raptors.


Assuntos
Criopreservação/veterinária , Águias/fisiologia , Ejaculação/fisiologia , Preservação do Sêmen/veterinária , Animais , Congelamento , Masculino , Preservação do Sêmen/métodos , Motilidade dos Espermatozoides , Espermatozoides/citologia , Espermatozoides/fisiologia
16.
PLoS One ; 8(3): e59189, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23544054

RESUMO

BACKGROUND: Sperm cryopreservation has become an indispensable tool in biology. Initially, studies were aimed towards the development of efficient freezing protocols in different species that would allow for an efficient storage of semen samples for long periods of time, ensuring its viability. Nowadays, it is widely known that an important individual component exists in the cryoresistance of semen, and efforts are aimed at identifying those sperm characteristics that may allow us to predict this cryoresistance. This knowledge would lead, ultimately, to the design of optimized freezing protocols for the sperm characteristics of each male. METHODOLOGY/PRINCIPAL FINDINGS: We have evaluated the changes that occur in the sperm head dimensions throughout the cryopreservation process. We have found three different patterns of response, each of one related to a different sperm quality at thawing. We have been able to characterize males based on these patterns. For each male, its pattern remained constant among different ejaculates. This latter would imply that males always respond in the same way to freezing, giving even more importance to this sperm feature. CONCLUSIONS/SIGNIFICANCE: Changes in the sperm head during cryopreservation process have resulted useful to identify the ability of semen of males for freezing. We suggest that analyses of these response patterns would represent an important tool to characterize the cryoresistance of males when implemented within breeding programs. We also propose follow-up experiments to examine the outcomes of the use of different freezing protocols depending on the pattern of response of males.


Assuntos
Criopreservação/métodos , Sêmen/citologia , Ovinos/metabolismo , Espermatócitos/citologia , Animais , Contagem de Células , Masculino , Cabeça do Espermatozoide
17.
Rev. cient. (Maracaibo) ; 19(2): 153-158, mar.-abr. 2009. tab
Artigo em Espanhol | LILACS | ID: lil-548714

RESUMO

Se utilizó el Análisis Automatizado de la Morfometría Espermática (ASMA) con el fin de determinar las dimensiones de la cabeza del espermatozoide (DCE) en semen de cerdos domésticos según la edad, además de agrupar las medidas obtenidas en subpoblaciones espermáticas (SP). Se evaluaron 36 muestras de semen fresco y diluido de 20 cerdos los cuales se clasificaron en dos categorías. A: menores de 18 meses de edad y B: mayores de 18 meses de edad. Las DCE (Largo, µm/ Ancho, µm/ Área, µm 2 y Perímetro, µm) se analizaron en frotis teñidos con Hemacolor ® mediante Sperm-Class Analyser ® (SCA) y los valores obtenidos guardados en una base de datos. El procedimiento GLM fue utilizado para evaluar el efecto de la edad del cerdo sobre las DCE y el análisis de agrupamiento (FASTCLUS) para identificar las SP. Los espermatozoides provenientes de cerdos mayores de 18 meses de edad presentaron mayor longitud (8,84 vs. 8,95 µm) que los cerdos menores de 18 meses de edad, sin embargo, las medias correspondientes al ancho (4,44 vs. 4,32 µm), área (33,33 vs. 32,39µm 2) y perímetro (27,65 vs. 26,3 µm) fueron más pequeñas en los cerdos de mayor edad. Dos SP fueron obtenidas con el fin de ratificar las diferencias observadas entre las 2 categorías de edades evaluadas (P<0,001). La población que incluyó los espermatozoides con las mayores dimensiones disminuyó de 41,61 por ciento en cerdos menores de 18 meses a 20,78 por ciento en cerdos mayores de 18 meses. Contrariamente, la SP que contenía los espermatozoides de menor tamaño incrementó de un 58,39 por ciento en cerdos menores de 18 meses a 79,22 por ciento en cerdos mayores de 18 meses. En conclusión, la edad de los cerdos influye significativamente sobre las DCE. Los cerdos de mayor edad tienen 20 por ciento más de espermatozoides de menor tamaño que los cerdos más jóvenes.


Assisted Sperm Morphometry Analysis (ASMA) was used to determine the sperm head dimensions (DCE) of boar by age, and then the data set clustered in sperm subpopulations (SP). To this purpose were evaluated 36 fresh and diluted semen samples of 20 Dalland domestic pigs, which were classified in 2 categories: under 18 months old and over 18 months old. The DCE (Length, µm/ Width, µm/, Area, µm 2 / and Perimeter, µm) were analyzed in slides stained by Hemacolor ® by the Sperm-Class Analyser ® (SCA), and the mean measurements recorded. A GLM procedure was performed to evaluate the effects of boar age on sperm head dimensions and clustering analysis (FAST-CLUS procedure) to separate in SP. Spermatozoa collected from older boar (over 18 months old) had head length larger (8.84 vs. 8.95 µm) than younger boar (under 18 months old), however, the width (4.44 vs. 4.32 µm), area (33.33 vs. 32.39 µm 2) and perimeter (27.65 to 26.3 µm) were smaller in older boar than younger boar. Two SP were clustered in this trial toratify the differences between younger and older pigs. The mean values of each DCE among the SP were significantly dif-ferent (P<0.001). Thus, the percentage of representation of the subpopulation that includes those spermatozoa whose dimensions are the largest decreased from 41.61 percent in pigs under 18 months old to 20.78 percent in pigs over 18 months old. Whereas, the percent of representation of the SP containing the smallest sper-matozoa increased from 58.39 percent in pigs under 18 months old to 153 79.22 percent in pigs over 18 months old. In conclusion, the age of sexually mature domestic male pig had a significant effect on the morphometric traits of their spermatozoa. Older boar had 20 percent more of smaller spermatozoa than younger boar.


Assuntos
Bovinos , Animais , Esfregaço Vaginal/veterinária , Contagem de Espermatozoides/veterinária , Cabeça do Espermatozoide , Sus scrofa/anatomia & histologia , Medicina Veterinária
18.
Reproduction ; 137(2): 225-35, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19028926

RESUMO

Fe(2)(+)/ascorbate, hydrogen peroxide (H(2)O(2)), and hypoxanthine/xanthine oxidase (XOD) are commonly used for inducing oxidative stress on spermatozoa. A comparative study of these agents was carried out on thawed spermatozoa from red deer. First, we tested a high, medium, and low concentration of each agent: 100, 10, and 1 microM Fe(2)(+) (hydroxyl radical generator); 1 mM, 100, and 10 microM H(2)O(2); and 100, 10, and 1 mU/ml XOD (superoxide and H(2)O(2) generator), incubated at 37 degrees C for 180 min. Intracellular reactive oxygen species (ROS; H(2)DCFDA) increased with dose and time similarly for the three systems at each concentration level. Motility and mitochondrial membrane potential (Deltapsi(m)) were considerably decreased by H(2)O(2) (1 mM and 100 microM) and XOD (100 and 10 mU/ml). Only 1 mM H(2)O(2) reduced viability. The antioxidant Trolox (10 microM) reduced intracellular ROS, but could not prevent the H(2)O(2) or XOD effects. In a second experiment, YO-PRO-1 and M540 were used as apoptotic and membrane stability markers respectively. Only H(2)O(2) increased the proportion of apoptotic and membrane-destabilized spermatozoa. Catalase added to XOD prevented Deltapsi(m) loss, confirming that H(2)O(2) was the causative agent, not superoxide. In a third experiment, caspase activation was tested using the (FAM-VAD-FMK) probe. Viable spermatozoa with activated caspases could be detected in untreated samples, and only H(2)O(2) increased their proportion after 60 min. There were important differences between ROS generators, H(2)O(2) being the most cytotoxic. Although H(2)O(2) and XOD caused Deltapsi(m) dissipation, this was not reflected in increasing apoptotic markers.


Assuntos
Cervos/metabolismo , Peróxido de Hidrogênio/farmacologia , Espécies Reativas de Oxigênio/farmacologia , Espermatozoides/citologia , Animais , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Biomarcadores/análise , Caspases/análise , Caspases/metabolismo , Catalase/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Ativação Enzimática , Citometria de Fluxo , Radical Hidroxila/farmacologia , Ferro/farmacologia , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Microscopia de Fluorescência , Estresse Oxidativo , Espécies Reativas de Oxigênio/análise , Motilidade dos Espermatozoides/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Espermatozoides/metabolismo , Superóxidos/farmacologia , Fatores de Tempo , Xantina Oxidase/farmacologia
19.
Rev. cient. (Maracaibo) ; 18(5): 570-577, sept.-oct. 2008. tab
Artigo em Espanhol | LILACS | ID: lil-548647

RESUMO

Para determinar los parámetros morfométricos de la cabeza espermática en semen porcino, así como evidenciar la presencia de subpoblaciones espermáticas fueron evaluadas 20 muestras seminales de 10 verracos Dalland. Sobre semen fresco y refrigerado fue evaluada la motilidad, vitalidad, acrosomas alterados y/o ausentes y anormalidades espermáticas. Mediante el análisis automatizado de la morfología espermática (ASMA), en frotis teñidos con Hemacolor®, se realizaron las mediciones de la cabeza espermática: Longitud (µm), Ancho (µm), Área (µm2), Perímetro (µm) y función Largo/Ancho. El efecto del proceso de refrigeración sobre las variables de calidad seminal y morfometría, se analizaron utilizando el GLM (SAS®) y para identificar las subpoblaciones espermáticas, se utilizó el procedimiento FASTCLUS (SAS®). La refrigeración a 16°C por 24 horas no afectó las características de calidad seminal de los eyaculados, pero si afectó las características morfométricas. La longitud de la cabeza disminuyó de 8,82 a 8,71 mm, así como el perímetro de 30,08 a 29,05 µm, mientras que aumentaron los valores de ancho (4,36 a 4,45 µm) y área (33,13 a 33,14 µm2). Se identificaron tres subpoblaciones espermáticas, con valores de distribución de 28,45 por ciento para la subpoblación 1 (espermatozoides grandes), 51,20 por ciento para la subpoblación 2 (medianos) y 20,35 por ciento para la subpoblación 3 (pequeños), las cuales se ven alteradas significativamente durante el proceso de refrigeración a 16°C.


To determine the morphometric parameters of the sperm head, and identify the presence of separate sperm subpopulations in boar semen were evaluated 20 ejaculate samples of 10 boars. Sperm motility, viability, acrosome integrity and morphological abnormalities were evaluated on fresh and cooling semen samples. By means Assisted Sperm Morphometry Analysis (ASMA), in slides stained by Hemacolor®, were determined the morphometric dimensions: Length (µm), Width (µm), Area (µm2), Perimeter (µm), and function Length/Width. Effect of cooling procedure on variables of semen quality and morphometric parameters were analyzed using GLM (SAS®). For identify the sperm subpopulations was used FASTCLUS procedure (SAS®). Cooling at 16°C for 24 hours did not affect the parameters of semen quality, but affected morphometric characteristics. Sperm head length decreased of 8.82 to 8.71 µm, and the sperm head perimeter of 30.08 to 29.05 µm, however, the width (from 4.36 to 4.45 mm) and area sperm head increased (33.13 to 33.14 µm2). Our results demonstrated that three separate sperm subpopulations coexist in boar ejaculates, 28.45% in the subpopulation 1 (larges), 51.20% in the subpopulation 2 (average), and 20.35% in the subpopulation 3 (small). These sperm subpopulation changed their distribution during cooling process.


Assuntos
Animais , Densidade Demográfica , Preservação do Sêmen/métodos , Cabeça do Espermatozoide , Suínos , Medicina Veterinária
20.
Rev. cient. (Maracaibo) ; 17(5): 508-513, sept.-oct. 2007. ilus, tab
Artigo em Inglês | LILACS | ID: lil-548544

RESUMO

El objetivo de este estudio fue determinar el efecto de la criopreservación sobre las características morfométricas de las cabezas de espermatozoides de toros Brahman y sus mestizos. Cinco eyaculados fueron colectados de 4 toros y diluidos a 30°C en una solución de leche descremada-yema de huevo. Por cada muestra se hicieron dos frotis: uno del semen diluido, antes de su congelación en vapores de nitrógeno líquido, y otro de semen descongelado una semana después de la congelación. Todos los frotis fueron secados al aire y coloreados con Hemacolor®. Se analizaron las dimensiones de la cabeza espermática para un mínimo de 150 espermatozoides por muestra mediante el Sperm Class Analyser® (SCA). El procedimiento GLM se realizó para evaluar el efecto de la criopreservación sobre las dimensiones morfométricas de las cabezas espermáticas. Las cabezas espermáticas de los toros fueron significativamente (P<0,001) menores en los espermatozoides criopreservados que en las muestras frescas para la longitud (9,00 µm vs. 9,43 µm), el ancho (4,82 µm vs. 5,13 µm), el perímetro (32,46 µm vs. 33,69 µm) y el área (36,20 µm²vs. 39,97 µm²) para todos los toros. Así mismo, se encontraron diferencias (P<0,001) de todos los parámetros morfométricos de los toros evaluados, encontrándose dimensiones de cabeza menores en las muestras descongeladas. La variabilidad individual (CV) de las medidas de cabeza espermática de los toros osciló entre el 5,9 y el 10,2 por ciento para las muestras frescas y descongeladas, respectivamente. En conclusión, este estudio indica que el proceso de criopreservación de semen de toro afecta la morfometría, al reducir las dimensiones de la cabeza espermática de toros Brahman y sus cruces. Las diferencias entre los toros evaluados puede ser indicativo de diferencias individuales al proceso de criopreservación.


The objective of this study was to determine the effect of cryopreservation on morphometrics characteristic of Brahman and their crossbred bull sperm heads. Five ejaculates were collected from 4 bulls and diluted at 30°C in a skim milk-egg yolk extender. Two microscope slides were prepared from single extended sperm samples prior to freezing in nitrogen vapors, and another one after thawing, sperm smears were prepared as described above. All slides were air dried and stained with Hemacolor®. Sperm-head dimensions for a minimum of 150 sperm heads/samples were analysed from each sample by means of the Sperm-Class Analyser® (SCA), and the mean measurements recorded. A GLM procedure was performed to evaluate the effect of cryopreservation on sperm head morphometric dimensions. Bull sperm heads were significantly (P<0.001) smaller in frozen-thawed spermatozoa than in the extended samples for length (9.00 µm vs. 9.43 µm), width (4.82 µm vs. 5.13 µm), perimeter (32.46 µm vs. 33.69 µm) and area (36.20 µm2 vs. 39.97 µm2) for all bulls. Also, differences (P<0.001) were found within all bulls for whole morphometric parameters. The individual variability of sperm head measurements across all bulls ranged from 5.9% to 10.2% for fresh and thawed samples, respectively. In conclusion, the present study indicate that cryopreservation of bull semen did affect the morphometry to reduce the dimensions of Brahman and crossbred bull sperm heads. The differences among bulls may be indicative of the individual bull resistance to the cryopreservation process.


Assuntos
Bovinos , Animais , Criopreservação/veterinária , Tamanho do Órgão , Cabeça do Espermatozoide , Medicina Veterinária
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