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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

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Thoroughly analyzing the sperm and exploring the information obtained using artificial intelligence (AI) could be the key to improving fertility estimation. Artificial neural networks have already been applied to calculate zootechnical indices in animals and predict fertility in humans. This method of estimating the results of reproductive biotechnologies, such as in vitro embryo production (IVEP) in cattle, could be valuable for livestock production. This study was developed to model IVEP estimates in Senepol animals based on various sperm attributes, through retrospective data from 290 IVEP routines performed using 38 commercial doses of semen from Senepol bulls. All sperm samples that had undergone the same procedure during sperm selection for in vitro fertilization were evaluated using a computer-assisted sperm analysis (CASA) system to define sperm subpopulations. Sperm morphology was also analyzed in a wet preparation, and the integrity of the plasma and acrosomal membranes, mitochondrial potential, oxidative status, and chromatin resistance were evaluated using flow cytometry. A previous study identified three sperm subpopulations in such samples and the information used in tandem with other sperm quality variables to perform an AI analysis. AI analysis generated models that estimated IVEP based on the season, donor, percentage of viable oocytes, and 18 other sperm predictor variables. The accuracy of the results obtained for the three best AI models for predicting the IVEP was 90.7, 75.3, and 79.6%, respectively. Therefore, applying this AI technique would enable the estimation of high or low embryo production for individual bulls based on the sperm analysis information.

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Sperm routinary fitness evaluation is not sufficient to predict bull reproductive capacity as they present differences in fertility up to 40%. Among the defects which compromise spermatozoa functionality, new approaches consider the study of sperm chromatin, which is the core structure containing paternal genetic information. Sperm chromatin needs to be compacted to maintain the integrity of DNA, which occurs by binding nucleoproteins with high affinity to DNA. In the last stages of sperm maturation, chromatin is hyper-compacted by basic proteins called protamines in a process named protamination. In this review, we summarized intrinsic and extrinsic factors that are suggested to influence protamination in bull spermatozoa, considering old and new evidence from human and murine spermatozoa. Also, the current approaches to evaluate bull protamination and its relationship with fertility were described. Nevertheless, the physiological mechanisms of protamination are still poorly understood.

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The association between advanced paternal age and impaired reproductive outcomes is still controversial. Several studies relate decrease in semen quality, impaired embryo/fetal development and offspring health to increased paternal age. However, some retrospective studies observed no alterations on both seminal status and reproductive outcomes in older men. Such inconsistency may be due to the influence of intrinsic and external factors, such as genetics, race, diet, social class, lifestyle and obvious ethical issues that may bias the assessment of reproductive status in humans. The use of the murine model enables prospective study and owes the establishment of homogeneous and controlled groups. This study aimed to evaluate the effect of paternal age on in vitro embryo development at 4.5 day post conception and on in vivo fetal development at 16 days of gestation. Murine females (2-4 months of age) were mated with young (4-6 months of age) or senile (18-24 months of age) males. We observed decreased in vitro cleavage, blastocyst, and embryo development rates; lighter and shorter fetuses in the senile compared to the young group. This study indicated that advanced paternal age negatively impacts subsequent embryo and fetal development.

Assuntos

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Sperm routinary fitness evaluation is not sufficient to predict bull reproductive capacity as they present differences in fertility up to 40%. Among the defects which compromise spermatozoa functionality, new approaches consider the study of sperm chromatin, which is the core structure containing paternal genetic information. Sperm chromatin needs to be compacted to maintain the integrity of DNA, which occurs by binding nucleoproteins with high affinity to DNA. In the last stages of sperm maturation, chromatin is hyper-compacted by basic proteins called protamines in a process named protamination. In this review, we summarized intrinsic and extrinsic factors that are suggested to influence protamination in bull spermatozoa, considering old and new evidence from human and murine spermatozoa. Also, the current approaches to evaluate bull protamination and its relationship with fertility were described. Nevertheless, the physiological mechanisms of protamination are still poorly understood.(AU)

Assuntos

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Spermatozoa have a spontaneous ability to take up exogenous DNA in a process regulated by specific mechanisms. This ability has been used to carry exogenous DNA into oocytes during fertilization to produce transgenic animals; a process called sperm-mediated gene transfer (SMGT). However, it is still an inefficient method and little is known about the effect of exogenous DNA once associated with spermatozoa, on sperm characteristics. Therefore, the objective of the present work was to evaluate the effects of exogenous DNA length and its amount on DNA uptake by bovine spermatozoa as well as spermatozoa viability. For that, spermatozoa (5 × 106 cells/mL) were incubated for 1 h at 38.5 °C with different exogenous DNA lengths (2.2, 5.5, or 8.5 kb) at different concentrations (number of molecules or ng). The association of exogenous DNA with spermatozoa was quantified by PCR real-time and the spermatozoa viability was evaluated by flow cytometry. Here, we show that no matter the amount of exogenous DNA used, larger sequences are less efficiently (p ˂ 0.05) associated with bovine spermatozoa. Besides that, the length and amount of exogenous DNA do not compromise sperm viability. Taken together, the results support that the length of exogenous DNA is more important than the amount used to influence its association with sperm cells. Thus, the size and quantity of exogenous DNA can be optimized to increase SMGT protocols, without altering the sperm viability.

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Although bovine embryo in vitro production (IVP) is a common assisted reproductive technology, critical points warrant further study, including sperm traits and oxidative status of sperm for in vitro fertilization (IVF). Our aim was to evaluate whether the lipid peroxidation index of commercial bull semen is influenced by sperm traits and oxidative status of sperm populations selected using Percoll® gradient. Semen straws from 48 batches from 14 Nelore bulls were thawed individually, analyzed for motility and subjected to Percoll selection. After Percoll, the lipid peroxidation index of the extender was evaluated, whereas selected sperm were analyzed for motility, acrosome and membrane integrity, mitochondrial membrane potential, chromatin resistance and oxidative potential under IVF conditions. Batches were divided retrospectively in four groups according to lipid peroxidation index. Sperm from Group 4 with the lowest index of lipid peroxidation had, after Percoll selection, greater plasma membrane integrity (81.3%; P = 0.004), higher mitochondrial potential (81.1%; P = 0.009) and lower oxidative potential (135.3 ng thiobarbituric acid reactive substances (TBARS)/ml; P = 0.026) compared with Group 1 with highest lipid peroxidation index (74.3%, 73% and 213.1 ng TBARS/ml, respectively). Furthermore, we observed negative correlations for the lipid peroxidation index with motility, membrane integrity and mitochondrial potential, and positive correlations with oxidative potential. In conclusion, oxidative stress in semen straws, as determined using lipid peroxidation in the extender, is associated with sperm traits and their oxidative potential under IVF conditions. These results provided further insights regarding the importance of preventing oxidative stress during semen handling and cryopreservation, as this could affect sperm selected for IVF. Finally, Percoll selection did not completely remove sperm with oxidative markers.

Assuntos

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The acetic acid-urea polyacrylamide gel electrophoresis system could separate very similar basic proteins on differences in size and effective charge. This system has been used for many years to analyse histones and their post-translational modifications and widely used in the study of mammal protamines. Two types of protamine have been described, the protamine 1 (P1) and the protamine 2 (P2) family members, which are synthetized by PRM1 and PRM2 genes. The ratio of P1 and P2 is important for predicting fertility in humans and mice. Therefore, the quantification of protamines is a fundamental step in order to establish the ratio between P1 and P2 in these species. In other mammals, studies linking sperm protamination and the protamine ratio with fertility are increasing. So, the use of an effective technique to separate and quantify protamines is important to study sperm P1/P2 ratio. Therefore, this article describes in detail a feasible and useful procedure to isolate bovine sperm protamines, to perform pre-electrophoresis with PEG solution and finally to carry out acid-urea polyacrylamide gel electrophoresis in reverse polarity. This technique allows a clear separation and efficient detection of bovine sperm protamines.

Assuntos

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This study aimed to evaluate the effect of seminal plasma on bovine sperm cryopreservation and to assess the integrity of plasma and acrosomal membranes, mitochondrial potential, remodelling of F-actin cytoskeleton and sperm chromatin fragmentation during the cooling, equilibrium and freezing/thawing stages. Six ejaculates collected from seven Nelore bulls (n = 42) were used in this study. Each ejaculate was divided into two aliquots (with seminal plasma = SP group; without seminal plasma = NSP group) and packed to a final concentration of 50 × 106 sperm per straw. Statistical analyses were performed using SAS software (version 9.3), and p ≤ .05 was considered significant. A time effect was observed for all sperm characteristics (p < .05), except for chromatin fragmentation (p > .05). The presence of seminal plasma better preserved the acrosomal integrity (SP = 75.2% and NSP = 71.7%; p < .05) and also provided lower F-actin remodelling during cryopreservation process (SP = 29.9% and NSP = 32.4%; p < .05). Regarding to the cryopreservation stages, it was observed that cooling step induced higher remodelling of F-actin than the equilibrium and freezing/thawing stages (56.3%, 32.2% and 23.9%, respectively; p < .05). The equilibrium step had minor influence on overall sperm characteristics while the freezing/thawing stage was responsible for the highest percentage of damage in plasma membrane (-65.2%), acrosomal membrane (-34.0%) and mitochondrial potential (-48.1%). On the other hand, none of the cryopreservation stages affected chromatin integrity. It was concluded that the presence of seminal plasma provides increased acrosomal integrity and reduced remodelling of F-actin cytoskeleton. Higher F-actin remodelling is observed after the cooling step while the freezing/thawing step is most damaging to sperm membranes and mitochondrial potential during bovine sperm cryopreservation.

Assuntos

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In vitro embryo production (IVP) efficiency is reduced when compared to in vivo. The basic knowledge of bovine in vitro oocyte maturation (IVM) mechanisms provides support to improve in vitro embryo production yields. The present study assessed the effects of bone morphogenetic protein 15 (BMP15), fibroblast growth factor 16 (FGF16) and their combined action on cumulus cells (CC) expansion, oocyte and CC DNA fragmentation, oocyte nuclear maturation, energetic metabolism and progesterone production in bovine IVM. Cumulus-oocyte complexes (COC) were matured in control or supplemented media containing BMP15 (100 ng/ml), FGF16 (10 ng/ml) or BMP15 combined with FGF16; and assessed at 0 and 22 hr of IVM. BMP15 alone or its association with FGF16 enhanced cumulus expansion. BMP15 decreased DNA fragmentation in both CC and oocytes, and improved oocyte nuclear maturation rate. In addition, BMP15 increased CC progesterone production, an effect not previously reported. The present study reinforces previous data pointing to a beneficial influence of BMP15 during IVM, while providing novel evidence that the underlying mechanisms involve increased progesterone production.

Assuntos