Chronological characterization of sperm morpho-functional damage and recovery after testicular heat stress in Nellore bulls.

Heat stress (HS) affects spermatogenesis and sperm maturation, decreasing sperm quality. Yet sperm morpho-functional changes caused by HS in Nellore bulls are not fully elucidated. This study aimed to show the chronological effects on sperm quality of HS during spermatogenesis and sperm maturation until recovery of the seminiferous epithelium in Nellore bulls. Nine Nellore bulls were distributed into control and heat stress (HS-scrotal bags/96 h) groups. The study was divided into five Periods: 1. Control (14-7 days before HS); 2. Stored sperm (0-7 days after HS); 3. Sperm maturation and late spermatogenesis (14-42 days after HS); 4. Early spermatogenesis (49-63 days after HS), and 5. Recovery (70-77 days after HS). Semen was collected once a week and evaluated for sperm motility, morphology, plasma, acrosome, and mitochondrial membranes, lipid peroxidation, and DNA fragmentation. Sperm characteristics were similar between groups in Periods 1 (control). During Period 2, HS increased detached normal head defect and decreased mitochondrial membrane potential, denoting effects on the sperm stored at the epididymis cauda. In Period 3, HS decreased sperm motility, plasma membrane integrity, and mitochondrial membrane potential and increased abnormal sperm, lipid peroxidation, and DNA fragmentation; reflecting the effects on sperm that were in the epididymis body and head and late spermatogenesis (spermiogenesis and meiosis). In Period 4, HS maintained a reduction in the mitochondrial membrane potential and an increase in abnormal sperm; injuries that could occur during early spermatogenesis (mitosis). Finally, in Period 5, the groups were similar, confirming the recovery of the seminiferous epithelium after HS. This study provides insights on the effects of HS on the complete process of sperm maturation and spermatogenesis, until recovery in sperm from Nellore bulls.

Sperm function and oxidative status: Effect on fertility in Bos taurus and Bos indicus bulls when semen is used for fixed-time artificial insemination.

Semen quality is one of the criteria used for the selection of bulls with relatively greater fertility. In addition, bull fertility depends on the integrity and function of all sperm structures. The aim of this study, therefore, was to determine associations when there was conducting of conventional and functional techniques for the evaluation of sperm samples from bulls with known fertility history as determined when semen from these bulls was used for fixed-time artificial insemination programs. The study was designed in a 2 × 2 factorial arrangement, with one factor being breed (Angus x Nellore) and the other fertility (greater x lesser). Greater fertility groups were composed of ten Angus and 11 Nellore bulls, while lesser fertility groups were composed of ten Angus and seven Nellore bulls. Sperm were analyzed, in four cryopreserved distinct batches for each animal, for morphology, kinetics, plasma and acrosomal membrane integrity, mitochondrial activity and mitochondrial membrane potential, DNA integrity and oxidative status. There was no difference in characteristics commonly used in sperm quality conventional analysis. The results from functional analysis indicated an important association between mitochondrial dysfunctions, oxidative stress, and damage to sperm structures in lesser fertility bulls. Greater fertility bulls had greater sperm quality and indicators of functional cell structures. The associations, when there were evaluations using different techniques, indicate the importance of evaluation and correlation between different sperm functions to understand effects of distinct parameters on sperm fertilization capacity.

Hydroxy-selenomethionine as an organic source of selenium in the diet improves boar reproductive performance in artificial insemination programs.

This study aimed to compare different selenium (Se) sources in the diet on boar's semen quality and fertility. For this, 28 boars aged 8 to 28 mo were fed with the following dietary treatments for 95 d: 0.3 mg Se/kg as sodium selenite (SS; n = 14) and 0.3 mg Se/kg as hydroxy-selenomethionine (OH-SeMet; n = 14). During this period, two experiments were carried out. In experiment 1, the semen of all boars was evaluated every 2 wk. Raw semen was initially evaluated for the processing of seminal doses, which were stored at 17 °C for 72 h, followed by sperm quality assessments. Furthermore, Se concentration and glutathione peroxidase (GPx) activity were measured in the seminal plasma. In experiment 2, 728 females were inseminated weekly with seminal doses from boars of the different experimental groups to further assess in vivo fertility and litter characteristics. Results demonstrated that boars fed OH-SeMet had more Se in their seminal plasma (P < 0.05), showing the greater bioavailability of the organic source in the male reproductive system. Moreover, boars fed OH-SeMet tended (P < 0.10) toward a higher total sperm count in the ejaculate (66.60 vs. 56.57 × 109 sperm) and the number of seminal doses (22.11 vs. 18.86; 3 × 109 sperm/dose) when compared with those fed SS. No effect of the dietary treatments was observed on GPx activity in seminal plasma (P > 0.05) as well as on raw and stored semen quality (P > 0.05). Under in vivo conditions, seminal doses from boars fed OH-SeMet tended (P < 0.10) toward a higher pregnancy rate at weeks 3, 5, and 8, and also resulted in a higher (P < 0.05) percentage of pregnant females in the overall period (99.30 vs. 97.00). In conclusion, the replacement of SS with OH-SeMet in boars' diet can improve sperm production and results in better reproductive performance for them, bringing greater productivity and profitability to artificial insemination centers and commercial pig farms.

The use of resveratrol decreases liquid-extend boar semen fertility, even in concentrations that do not alter semen quality.

In vitro and in vivo assays were conducted to investigate the effects of trans-resveratrol (RVT) on liquid-extended boar semen during 72 h of storage at 17 °C. Thirty-six ejaculates were collected from six boars, evaluated, and extended. RVT was then added at the indicated treatment concentration (0, 0.01, 0.1 or 1 mM), and the ejaculates were cooled to 17 °C and evaluated at 0, 24, 48, and 72 h. Samples were evaluated for sperm motility, kinetics, plasma and acrosome integrity, mitochondrial membrane potential, anion superoxide levels, lipoperoxidation, and antioxidant enzyme activity. In the follow-up experiment, twenty-eight gilts were fixed-time inseminated with 0 or 0.01 mM RVT liquid-extended boar semen. After five days, they were slaughtered, and their reproductive tracts were recovered. The embryos were collected, and the pregnancy, fertility, and viable embryo rates were calculated. In the in vitro assays, total motility, plasma and acrosome membrane integrity, mitochondrial membrane potential, anion superoxide levels, and lipoperoxidation did not change at any of the evaluation times with the use of RVT up to 0.01 mM. RVT decreased SOD activity without changes in GPx. RVT used at 1 mM showed harmful effects for almost all evaluated parameters. For the in vivo assay, the same pregnancy and fertility rates were observed for both groups, while the viable embryo rate was three-fold lower in the 0.01 mM group than in the 0 mM group. The results showed a dichotomous effect of RVT; a low concentration was not harmful in vitro but was catastrophic for embryo viability.

Reproductive parameters of Bos taurus and Bos indicus bulls during different seasons in tropical conditions: focus on an alternative approach to testicular assessments using ultrasonography.

The aim of the present study was to evaluate the reproductive status of Bos taurus and Bos indicus bulls during different seasons when bulls were in a tropical environment focusing on systematic assessment of the testes using B-mode ultrasonography and ImageJ software to evaluate the testicular parenchymal tissues. The experimental design is a 2 × 2 factorial arrangement, with breed (Bos Taurus - Simmental; Bos indicus - Nellore) and season (summer; winter) as factors. Testicular ultrasonic evaluation and conventional semen analysis were performed. The Simmental bulls had more major sperm defects than Nellore bulls in the summer (P = 0.0001). Additionally, Simmental bulls had a greater percentage of major sperm defects during the summer than winter months P = 0.045). Furthermore, Nellore bulls had a greater testicular parenchyma echogenicity, with a greater grayscale, (caudal parenchyma, P = 0.0155; cranial parenchyma, P = 0.001) and mediastinum grayscale than Simmental bulls (Nellore = 52.32 ± 03.60; Simmental = 35.72 ± 03.67; P = 0.003). Simmental bulls also had a greater testicular lesion area (P = 0.0147). Results indicated there was susceptibility to heat stress when Simmental bulls were maintained in tropical regions. The results of the present study indicate there is an association between results when there was use of conventional andrological evaluation procedures and results from ultrasonic analysis using ImageJ software that allows for earlier identification of tissue aberrations that could lead to impaired semen quality and fertility.

Sperm evaluation and morphological description of male genitalia of meerkats (Suricata suricatta).

In ex situ conditions, little is known about the reproductive biology of meerkats. The aim of present study was to describe the morphological aspects of male genital organs and accessory glands using macroscopic evaluation, ultrasonography, and radiography, as well as describing semen characteristics post-electroejaculation. The results indicated anatomical characteristics of meerkats are very similar to those of cats, having a prostate, accessory bulbourethral glands, and an elongated and radiopaque structure in the penis, which is indicative of there being a baculum. The testicular volume was 0.81 cm³ (± 0.10) and the relative testis weight was 1.37 cm³/kg (± 0.15). Both testicles are present in the scrotum, which has an ellipsoidal shape, homogeneous texture, hypoechoic parenchyma and are encased in a hyperechoic tunica albuginea. Electroejaculation was effectively induced in all animals for semen collection with utilization of medetomidine and ketamine. The values semen samples variables were as follows for volume - 0.125 ±â€¯0.193 mL, motility - 19.8 ±â€¯18.6 %, vigor - 1.9 ±â€¯1.0, concentration - 40.5 ±â€¯25.2 × 106 sperm/mL and morphologically normal sperm - 10.8 ±â€¯6.6 %. This is the first study in which there is a description of morphological and imaging aspects of the male reproductive tracts of meerkats, as well as the seminal characteristics after using electroejaculation for semen collection. Knowledge of anatomical and seminal characteristics is essential for implementation of assisted reproduction programs, as well as reproductive management in the species.

Practical methods to assess the effects of heat stress on the quality of frozen-thawed Belgian Blue semen in field conditions.

The increased exportation of semen and embryos of double-muscled beef breeds to tropical and developing countries makes it important to investigate the reproductive capacity of these breeds in adapting to tropical conditions. The aim of this study was to evaluate the quality of Belgian Blue semen collected after there is heat-stress (HS; as a mimic of tropical condition) compared with non-heat stressed (NHS; as their comfort zone), using practical spermatozoa staining methods such that prevail in developing countries. There was screening of semen kinetics using CASA and evaluation of their DNA-, acrosome, plasma membrane-integrity, and mitochondrial activity. For each staining technique, there was evaluation of 12 frozen-thawed semen samples from six Belgian Blue bulls collected after there were HS and NHS conditions in Belgium. Mixed linear regression models were used to assess the effects of HS for each CASA variable and staining method outcome using the replicate nested with bull as a random effect. There were differences (P < 0.05) in values when there were semen collections following HS and NHS conditions for several post-thawing kinetic variables. Furthermore, the mean percentages of DNA-, acrosome-, and plasma membrane-integrity, as well as mitochondrial activity were greater (P < 0.05) when semen was collected following NHS compared with HS conditions. Conclusively, results indicated that when there was collection of semen following HS conditions, there were detrimental effects on the viability and quality of Belgian Blue semen which is an important consideration for the semen collection, processing, and evaluation in tropical countries.

Heat stress effects on bovine sperm cells: a chronological approach to early findings.

Testicular heat stress affects sperm quality and fertility. However, the chronology of these effects is not yet fully understood. This study aimed to establish the early sequential effects of heat stress in bull sperm quality. Semen and blood samples of Nellore breed bulls were collected and distributed into control and testicular heat stress (scrotal bags/96 h) groups. Semen samples were evaluated for sperm motility, abnormalities, plasma membrane integrity, acrosomal membrane integrity, mitochondrial membrane potential, sperm lipid peroxidation, seminal plasma lipid peroxidation, and DNA fragmentation. Blood plasma was also evaluated for lipid peroxidation. An increase in sperm abnormalities was observed 7 days following heat stress. After 14 days, sperm lipid peroxidation increased and mitochondrial membrane function, sperm motility, and plasma membrane integrity decreased. Heat stress effects were still observed after 21 days following heat stress. An increase in sperm DNA fragmentation was observed as a late effect after 28 days. Thus, the initial effects of heat stress (i.e., increasing sperm abnormalities and lipid peroxidation) suggest the presence of oxidative stress in the semen that alters mitochondrial function, sperm motility, plasma membrane integrity, and belatedly, DNA fragmentation. Although sperm abnormalities persisted and increased over time, sperm lipid peroxidation, in turn, increased only until 21 days after heat stress. In this regard, these findings provide a greater understanding of the chronological effects of experimentally induced heat stress on bovine sperm, providing valuable insights about spermatogenesis during the first 28 days following heat stress.

Effect of senescence on morphological, functional and oxidative features of fresh and cryopreserved canine sperm.

The present research aimed to compare the hormonal profile, sperm quality and freezability of young and senile dogs. Dogs were assigned into Young Group (n = 11) and Senile Group (n = 11), additionally divided into Fresh Semen Group and Cryopreserved Semen Group. Males were evaluated for libido score and blood estrogen and testosterone assay. Sperm morphofunctional evaluations were performed based on Computer Assisted Sperm Analysis, morphology, mitochondrial activity, mitochondrial membrane potential, plasma and acrosomal membrane integrity, and DNA fragmentation. Sperm oxidative features were: protein oxidation, lipid peroxidation and production of advanced glycation end-products. Young dogs had higher libido score, sperm velocity average pathway, linearity of motility and mitochondrial activity index and lower percentage of major defects, total defects and proximal cytoplasmic droplet, despite the lack of difference between hormone profile of aged dogs. Fresh semen of senile dogs had increased percentage of spermatozoa with high mitochondrial membrane potential compared to young dogs and to cryopreserved sperm. Cryopreserved semen of young dogs had higher acrosomal membrane integrity compared to the Senile Group. In conclusion, sperm of aged dogs have reduced quality, signaled by higher morphological defects, ultimately altering sperm mitochondrial function and sperm kinetics. Furthermore, spermatozoa from senile dogs are more sensible to cryoinjury.

Field-testing a single-dose immunocontraceptive in free-ranging male capybara (Hydrochoerus hydrochaeris): Evaluation of effects on reproductive physiology, secondary sexual characteristics, and agonistic behavior.

Controlling wildlife populations to mitigate human-wildlife conflicts and the spread of zoonotic diseases is an ever-growing necessity. The objective of this study was to evaluate a single-dose anti-gonadotropin-releasing hormone vaccine (GonaCon, USDA/NWRC, Fort Collins, CO, USA) as a non-lethal alternative for population control in free-ranging, synanthropic male capybara. In addition to infertility efficacy of this treatment, potential effects on the alpha male's secondary sexual characteristics and agonist behavior need to be assessed because any alterations in these factors could lead to population management failure. The treatment group (n = 3) received 1 mL of the anti-GnRH vaccine, intramuscularly, and the control group (n = 2) a 1 mL sham vaccine. Reproductive behavior and social group dynamics were monitored for 30 days prior to inoculation (June 2017) with continuous observations occurring during the study period. Antifertility effects were assessed by conducting exams of testicular morphology, semen characteristics, and histological analysis (after 270 days via hemi-gonadectomy). Compared to the control group, the testicles of the treated males had severe atrophy (P <  0.05), oligozoospermia and greater numbers of sperm cells in a static developmental phase. Courtship and agonistic alpha male behavior were not altered, and the group's social integrity was maintained. Results indicate there was 100% infertility in capybara males, observed throughout the study period of 18 months, and equally important, the male's alpha characteristics were not affected by the treatment, which is imperative for successful capybara population control efforts.

Carnosine as malondialdehyde scavenger in stallion seminal plasma and its role in sperm function and oxidative status.

Semen biotechniques may impair sperm quality due to excessive production of reactive oxygen species (ROS). Additionally, products of the oxidative reaction, especially involving lipids (e.g., malondialdehyde - MDA), may be even more harmful to sperm. Carnosine, previously reported to be present in seminal plasma of several species, may be a key factor on sperm tolerance to biotechniques by counterattacking the deleterious influence of MDA. Therefore, the aim of this study was to measure the levels of carnosine present in equine seminal plasma and relate these findings with sperm function and oxidative status during cooling and cryopreservation. Thus, semen samples were collected from 40 stallions in duplicate (N = 80) and then submitted to cooling and cryopreservation. Samples were then allocated into groups of high and low tolerance to refrigeration and cryopreservation (bad cooler and good cooler/bad freezer and good freezer, respectively), and in groups of different concentrations of carnosine (High, Medium-high, Medium-low and Low carnosine). Samples were evaluated for sperm kinetics patterns, function of sperm structures and oxidative status. In good cooler samples, it was observed higher concentrations of carnosine (Good cooler: 224.98 ±â€¯19.16 ng/mL; Bad cooler: 159.72 ±â€¯15.99 ng/mL; p = 0.0056), ROS production (Good cooler: 26.40 ±â€¯18.33%; Bad cooler: 18.33 ±â€¯1.84%; p = 0.001) and lipid peroxidation rates (Good cooler: 193.23 ±â€¯18.22 ng/mL; Bad cooler: 131.92 ±â€¯12.25; p = 0.0064). Groups of samples with higher carnosine concentrations had lower levels of malondialdehyde (High: 79.33 ±â€¯6.72 ng/mL; Medium-high: 140.45 ±â€¯11.70 ng/mL; Medium-low: 202.57 ±â€¯16.30 ng/mL and Low: 231.02 ±â€¯32.35 ng/mL; p < 0.05), demonstrating that carnosine was effective in removing lipid peroxidation products. Due to the removal of seminal plasma during the cryopreservation process, no differences occurred in carnosine levels between bad and good freezer groups. In this context, this study provides relevant data for future therapies using carnosine during cryopreservation, aiming to replace the levels lost due to the necessary removal of seminal plasma.

Induced sperm oxidative stress in dogs: Susceptibility against different reactive oxygen species and protective role of seminal plasma.

Oxidative stress (OS) is characterized by an unbalance between increased levels of reactive oxygen species (ROS) and/or impaired antioxidant protection. In this context, the composition of seminal plasma (SP) plays a key role in protecting sperm against OS. However, reproductive biotechnologies applied to dogs recommend the removal of SP. Thus, antioxidant therapy may be an important alternative when applying biotechniques such as semen cryopreservation in this specie. However, in order to be efficient, the choice of the ideal antioxidant in each condition is essential since each ROS is preferably neutralized by different antioxidant systems. Therefore, this study aims to evaluate the susceptibility of canine spermatozoa to different oxidative challenges (superoxide anion [O2-], hydrogen peroxide [H2O2], hydroxyl radical [OH-] and malondialdehyde [MDA]) in the present or absence of SP. We used ejaculates of eight dogs and submitted to induce oxidative challenges (with or without SP). After incubations, samples were evaluated for the susceptibility to lipid peroxidation, motility, mitochondrial activity and function, DNA integrity, plasma membrane and acrosome integrity. Sperm with SP had mitochondrial function preserved against ROS. However, in the absence of SP, H2O2 reduced mitochondrial membrane potential. In addition, regardless on SP, H2O2 was deleterious to sperm kinetics and plasma/acrosomal membranes. Incubation with OH- reduced mitochondrial activity and increased DNA fragmentation also independent on the absence of presence of SP. Furthermore, samples with SP were more resistant to lipid peroxidation (i.e., decreased concentration of TBARS). In conclusion, H2O2 and OH- appears to be the most deleterious ROS to dog sperm and SP protects the spermatozoa against mitochondrial injuries and lipid peroxidation.