RESUMO
This study investigated the impact of various Ge132 (Bis-carboxyethyl germanium sesquioxide) concentrations on frozen bovine semen. Ejaculates from three bulls were pooled and divided into six groups, each one with different Ge132 concentrations (0, 500, and 1000 µg/mL) and each group was incubated in different conditions (33°C for 30 min (D: D0, D500, and D1000), and the other was immediately cooled to 4°C (R: R0-control; R500 and R1000)). Thawed semen was evaluated for sperm characteristics by CASA and flow cytometer. Results showed better motility in the immediate cooling group without Ge132 compared with high Ge132 concentrations. Values for total motility dropped after 5 and 60 min in groups with high Ge132 levels and some control groups. Linearity increased with 1000 µg/mL Ge132, while straightness differed between moments in multiple groups. Membrane integrity was higher in a control group and certain Ge132 groups. Lower O2 - generation occurred without Ge132. After oxidative stress induction, lipid peroxidation intensity increased with arachidonic acid, but D1000 had lower peroxidation than R0. Overall, Ge132 appears to have provided protection against PLM when subjected to oxidative stress, since even at high concentrations it maintained sperm metabolism.
Assuntos
Antioxidantes , Criopreservação , Preservação do Sêmen , Motilidade dos Espermatozoides , Espermatozoides , Animais , Masculino , Bovinos , Criopreservação/veterinária , Criopreservação/métodos , Preservação do Sêmen/veterinária , Preservação do Sêmen/métodos , Espermatozoides/efeitos dos fármacos , Antioxidantes/farmacologia , Motilidade dos Espermatozoides/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Crioprotetores/farmacologia , Peroxidação de Lipídeos/efeitos dos fármacos , Germânio/farmacologia , Sêmen/efeitos dos fármacos , Análise do Sêmen/veterináriaRESUMO
In recent decades, there has been a growing interest in optimizing the protocols intended to sperm cryopreservation in domestic animals. These protocols include initial cooling, freezing, and thawing. While different attempts have been devised to improve sperm cryopreservation, the efficiency of this reproductive biotechnology is still far from being optimal. Furthermore, while much attention in improving cooling/freezing, less emphasis has been made in how thawing can be ameliorated. Despite this, the conditions through which, upon thawing, sperm return to physiological temperatures are much relevant, given that these cells must travel throughout the female genital tract until they reach the utero-tubal junction. Moreover, the composition of the media used for artificial insemination (AI) may also affect sperm survival, which is again something that one should bear because of the long journey that sperm must make. Furthermore, sperm quality and functionality decrease dramatically during post-thawing incubation time. Added to that, the deposition of the thawed sperm suspension devoid of seminal plasma in some species during an AI is accompanied by a leukocyte migration to the uterine lumen and with it the activation of immune mechanisms. Because few reviews have focused on the evidence gathered after sperm thawing, the present one aims to compile and discuss the available information concerning ruminants, pigs and horses.
Assuntos
Animais Domésticos , Criopreservação , Inseminação Artificial , Preservação do Sêmen , Animais , Criopreservação/veterinária , Criopreservação/métodos , Preservação do Sêmen/veterinária , Preservação do Sêmen/métodos , Masculino , Inseminação Artificial/veterinária , Espermatozoides/fisiologia , Temperatura , Crioprotetores/farmacologia , Fatores de Tempo , Cavalos/fisiologiaRESUMO
After ejaculation, mammalian sperm undergo a series of molecular events conducive to the acquisition of fertilizing competence. These events are collectively known as capacitation and involve acrosomal responsiveness and a vigorous sperm motility called hyperactivation. When mimicked in the laboratory, capacitating bovine sperm medium contains bicarbonate, calcium, albumin and heparin, among other components. In this study, we aimed at establishing a new capacitation protocol for bovine sperm, using calcium ionophore. Similar to our findings using mouse sperm, bovine sperm treated with Ca2+ ionophore A23187 were quickly immobilized. However, these sperm initiated capacitation after ionophore removal in fresh medium without heparin, and independent of the Protein Kinase A. When A23187-treated sperm were used on in vitro fertilization (IVF) procedures without heparin, eggs showed cleavage rates similar to standardized IVF protocols using heparin containg synthetic oviduct fluid (IVF-SOF). However, when A23187 pre-treated sperm were further used for inseminating eggs in complete IVF-SOF-heparin, a significantly higher percentage of embryo development was observed, suggesting a synergism between two different signaling pathways during bovine sperm capacitation. These results have the potential to improve current protocols for bovine IVF that could also be applied in other species of commercial interest.
Assuntos
Calcimicina , Ionóforos de Cálcio , Criopreservação , Fertilização in vitro , Preservação do Sêmen , Capacitação Espermática , Espermatozoides , Animais , Bovinos , Masculino , Ionóforos de Cálcio/farmacologia , Criopreservação/veterinária , Criopreservação/métodos , Fertilização in vitro/veterinária , Fertilização in vitro/métodos , Calcimicina/farmacologia , Espermatozoides/efeitos dos fármacos , Espermatozoides/fisiologia , Capacitação Espermática/efeitos dos fármacos , Preservação do Sêmen/veterinária , Preservação do Sêmen/métodos , Feminino , Técnicas de Cultura Embrionária/veterinária , Desenvolvimento Embrionário/efeitos dos fármacosRESUMO
Long-term preservation of gametes has been identified as a tool to improve broodstock management and increase the number of juveniles produced by artificial fertilization. Paralichthys orbignyanus is an important commercial and recreational species distributed in marine and estuarine waters from Rio de Janeiro (Brazil) to the San Matías Gulf (Argentina). This work focused on studying the seminal quality of tank-reared P. orbignyanus, demonstrating that males are fluent year-round, with the highest yields at the early reproductive season. Fresh sperm exhibited good forward swimming, and samples could be refrigerated up to 48 h while retaining their motility after activation. The optimal conditions for P. orbignyanus sperm motility activation were established as 950 mOsmol/Kg and pH values between 7 and 7.9. Additionally, a well-defined protocol for semen vitrification was developed to assess the cryotolerance of this species' sperm. We successfully produced high-quality sperm samples, using two vitrification formulations containing trehalose and both z-1000 and x-1000 polymers, that can be used in a near-future in vitro embryo production program.
Assuntos
Criopreservação , Linguado , Estações do Ano , Preservação do Sêmen , Animais , Masculino , Preservação do Sêmen/veterinária , Preservação do Sêmen/métodos , Linguado/fisiologia , Criopreservação/veterinária , Criopreservação/métodos , Análise do Sêmen/veterinária , Espermatozoides/fisiologia , Sêmen/fisiologia , Vitrificação , Motilidade dos EspermatozoidesRESUMO
The study investigated midpiece defects in sperm from a 5-year-old Brangus bull with a high rate of semen batch rejection, due to morphologically abnormal sperm, with no reduction in sperm kinematics. A comprehensive evaluation was conducted over a 16-month period, involving 28 ejaculates. Notably, despite the high proportion of midpiece defects (average 37.73%, from 3% to 58%), the study revealed stable sperm production, with no discernible differences in the kinematic data before and after cryopreservation. Electron microscopy identified discontinuities in the mitochondrial sheath, characteristic of midpiece aplasia (MPA). The anomalies were attributed to be of genetic origin, as other predisposing factors were absent. Additionally, the electron microscopy unveiled plasma membrane defects, vacuoles and chromatin decondensation, consistent with previous findings linking acrosome abnormalities with midpiece defects. The findings underscored the necessity of conducting thorough laboratory evaluations before releasing cryopreserved semen for commercialization. Despite substantial morphological alterations, the initial semen evaluation data indicated acceptable levels of sperm kinematics, emphasizing the resilience of sperm production to severe morphological changes. This case report serves as a contribution to the understanding of midpiece defects in bull sperm, emphasizing the need for meticulous evaluation and quality control in semen processing and commercialization.
Assuntos
Criopreservação , Análise do Sêmen , Preservação do Sêmen , Espermatozoides , Masculino , Animais , Criopreservação/veterinária , Bovinos , Preservação do Sêmen/veterinária , Análise do Sêmen/veterinária , Espermatozoides/anormalidades , Espermatozoides/fisiologia , Fenômenos Biomecânicos , Peça Intermédia do Espermatozoide , Motilidade dos Espermatozoides , AcrossomoRESUMO
There are more than 200 species and subspecies of Neotropical Primates of which more than 40% are listed as threatened by the IUCN Red List of Threatened Species. Both in situ and ex situ conservation programs can benefit from the use of assisted reproductive technologies. The objective of this study was to evaluate, for the first time, cryopreservation techniques for Alouatta caraya semen. Semen samples were collected from five adult males, analyzed, and frozen in either Test-egg yolk or Test-soy lecithin-based extenders containing either 3 or 4% glycerol. Frozen-thawed samples were analyzed at 10, 40, and 80 min post-thaw. Egg yolk-based extenders were overall better than soy lecithin-based extenders. There was no significant difference between 3 and 4% glycerol in any of the parameters analyzed, however, 4% glycerol in egg yolk-based extender produced more favorable results for total motility, intact plasma membrane, lipid peroxidation, and DNA fragmentation index. This study brought novel information on semen characteristics and cryopreservation aspects for A. caraya, which can help shape future experiments to improve the outcome of frozen-thawed sperm for this and other species of Neotropical primates.
Assuntos
Alouatta , Criopreservação , Crioprotetores , Gema de Ovo , Preservação do Sêmen , Espermatozoides , Animais , Masculino , Criopreservação/veterinária , Criopreservação/métodos , Preservação do Sêmen/veterinária , Preservação do Sêmen/métodos , Gema de Ovo/química , Espermatozoides/fisiologia , Alouatta/fisiologia , Lecitinas , Glycine max/química , Glicerol , Motilidade dos Espermatozoides/efeitos dos fármacosRESUMO
This study aims to evaluate the effects of adding alpha lipoic acid (ALA) to the in vitro ovarian tissue culture medium, either fresh or after vitrification/warming. For this purpose, 10 ovaries from five adult sheep were used. Each pair of ovaries gave rise to 16 fragments and were randomly distributed into two groups: fresh (n = 8) and vitrified (n = 8). Two fresh fragments were fixed immediately and considered the control, while another six were cultured in vitro for 14 days in the absence; presence of a constant (100 µM/0-14 day) or dynamic (50 µM/day 0-7 and 100 µM/day 8-14) concentration of ALA. As for the vitrified fragments, two were fixed and the other six were cultured in vitro under the same conditions described for the fresh group. All the fragments were subjected to morphological evaluation, follicular development and stromal density (classical histology), DNA fragmentation (TUNEL), senescence (Sudan Black), fibrosis (Masson's Trichome), and endoplasmic reticulum stress (immunofluorescence). Measurements of the antioxidant capacity against the free radicals 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) and estradiol (E2) levels in the culture medium was performed. The results showed that in the absence of ALA, in vitro culture of vitrified ovarian fragments showed a significant reduction (P < 0.05) in follicular morphology and increased the presence of senescence and tissue fibrosis (P < 0.05). Dynamic ALA maintained E2 levels unchanged (P > 0.05) until the end of vitrified ovarian tissue culture and controlled the levels of ABTS and DPPH radicals in fresh or vitrified cultures. Therefore, it is concluded that ALA should be added to the vitrified ovarian tissue in vitro culture medium to reduce the damage that leads to loss of ovarian function. To ensure steroidogenesis during in vitro culture, ALA should be added dynamically (different concentrations throughout culture).
Assuntos
Ácido Tióctico , Técnicas de Cultura de Tecidos , Animais , Feminino , Ácido Tióctico/farmacologia , Ovinos , Técnicas de Cultura de Tecidos/veterinária , Ovário/efeitos dos fármacos , Folículo Ovariano/efeitos dos fármacos , Antioxidantes/farmacologia , Vitrificação , Criopreservação/veterináriaRESUMO
Some synthetic polymers can be used at low concentrations to reduce the toxicity of conventional cryoprotectant agents. In this study we investigated whether the addition of synthetic polymers to a conventional cryoprotectant solution would improve the cryopreservation of bovine ovarian tissue. Freshly collected ovaries from ten adult crossbred cows were incised using a scalpel in the frontal section. From each cow, ovarian cortical slices of 1 mm thickness were divided into 30 fragments of 3 × 3 mm, of which 10 served as fresh controls, 10 were vitrified with conventional cryoprotectant agents (2.93 M glycerol, 27 % w/v; 4.35 M ethylene glycol, 27 % w/v), and 10 were vitrified using the same cryoprotectant agents in addition to synthetic polymers (0.2 % PVP K-12, 0.2 % SuperCool X-1000 ™ w/v and 0.4 % SuperCool Z-1000 ™ w/v). After warming, histology was used to assess follicular quantity and integrity, while in vitro culture of mechanically isolated follicles encapsulated in an alginate matrix was performed for 15 days to assess their growth and hormonal production. Vitrified ovarian tissues presented abnormal morphology, a higher percentage of atretic follicles, and their isolated follicles had lower survival rates and lower frequency of antrum formation during in vitro culture compared to those from fresh tissue. At the end of culture, the follicles that had been cryopreserved produced less estradiol and progesterone than the fresh ones. The addition of synthetic polymers during tissue vitrification did not modify any of these parameters. We conclude that, under the conditions of this study, the use of this combination of synthetic polymers for tissue vitrification did not enhance the preservation of the morphological or functional integrity of bovine ovarian follicles.
Assuntos
Criopreservação , Crioprotetores , Etilenoglicol , Glicerol , Folículo Ovariano , Ovário , Vitrificação , Animais , Feminino , Bovinos , Crioprotetores/farmacologia , Criopreservação/métodos , Criopreservação/veterinária , Ovário/efeitos dos fármacos , Glicerol/farmacologia , Etilenoglicol/farmacologia , Folículo Ovariano/efeitos dos fármacos , Polímeros/farmacologia , Polímeros/química , Progesterona/farmacologia , Estradiol/farmacologia , Alginatos/química , Alginatos/farmacologiaRESUMO
The aim of this study was to assess the addition of 2% sodium caseinate in a commercial egg yolk-based medium in frozen ovine semen. Eight Dorper males were used for the study. The ejaculate was divided into two portions and frozen without (G1) or with the addition of 2% sodium caseinate (G2). Kinetic parameters were evaluated using CASA (computer-assisted sperm analysis), and membrane and acrosome integrity as well as oxidative stress were assessed using flow cytometry. After thawing, a thermoresistance test was conducted at time points T0 and T90. For the fertility test, 100 ewes were inseminated with semen from two rams selected based on in vitro parameters, one with good post-thaw quality (+70% total motility) and the other with low post-thaw quality (-55% total motility). For the fertility test, the females were divided into 4 groups for insemination: low-quality ram without caseinate (GBS = 25) and with caseinate (GBC = 25), and high-quality ram without caseinate (GAS = 25) and with caseinate (GAC = 25). Regarding the results of sperm kinetics, there was a statistically significant difference in the parameters of average path velocity (VAP) and curvilinear velocity (VCL) between the group frozen with BotuBov and the group with added caseinate. At time point T90, straight-line velocity maintained a trend (p < .06), with BotuBov® (BB group) being superior to caseinate this time, and in the linearity parameter, caseinate was superior to BotuBov®. Flow cytometry analysis showed no difference between any of the evaluated tests. In the fertility test, there was no statistically significant difference in the pregnancy rate between the BotuBOV® group (23%, 11/48) and the sodium caseinate group (BC group) (33%, 17/52), and no differences were observed in the male versus diluent interaction (p = .70). In conclusion, sodium caseinate supplementation did not influence sperm kinetic parameters and the fertility of sheep.
Assuntos
Caseínas , Criopreservação , Inseminação Artificial , Análise do Sêmen , Preservação do Sêmen , Motilidade dos Espermatozoides , Animais , Preservação do Sêmen/veterinária , Preservação do Sêmen/métodos , Masculino , Feminino , Criopreservação/veterinária , Criopreservação/métodos , Inseminação Artificial/veterinária , Caseínas/farmacologia , Análise do Sêmen/veterinária , Gravidez , Motilidade dos Espermatozoides/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Espermatozoides/fisiologia , Crioprotetores/farmacologia , Sêmen/efeitos dos fármacos , Fertilidade/efeitos dos fármacos , Ovinos , Carneiro DomésticoRESUMO
While cryopreservation of cauda epididymal sperm (SpCau) allows the preservation of post-mortem bulls' gametes, the process triggers sperm damage. Although improving post-thaw sperm quality, using egg yolk extenders (EY) raises biosafety concerns which forces the use of EY-free extenders (EYFE). Since EYFE are less efficient in preserving post-thaw sperm quality, a strategy for ejaculated sperm (SpEj) frozen with EYFE is to add an Equilibrium Time (ET) step period to the cryopreservation process. However, the ET effect on the quality of SpCau cryopreserved in EYFE remains unknown. Distinct from SpEJ, SpCau physiologically displays cytoplasmic droplets (CDs) in the flagellum that may benefit cell exchange during ET. We hypothesized that using ET in SpCau cryopreserved with EYFE impacts sperm morphofunctional features, CD area, and in vitro fertility ability. Extender nanoparticles were also assessed. Following collection from the cauda epididymis of six Nellore bulls by retrograde flow, SpCau were cryopreserved in EYFE BoviFree® (Minitube, Germany) using three ET protocols: ET0 (no-ET); ET2.5 (2.5 h-ET); and ET5 (5 h-ET). SpCau from ET2.5 and ET5 showed a higher (P ≤ 0.05) percentage of motility and integrity of plasma and acrosome membranes and a smaller (P ≤ 0.05) distal CD area. There are no differences in sperm abnormalities, oxidative stress, capacitation-like events, and in vitro fertility ability. However, a better sperm recovery was found after Percoll® selection for ET2.5 and ET5. Interestingly, the number of nanoparticles in the extender decreased in post-thawed samples. In conclusion, an ET of 2.5 or 5 h is required for an efficient SpCau cryopreservation using an EYFE.