Lipid peroxidation in bull semen influences sperm traits and oxidative potential of Percoll®-selected sperm.

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.

An improved acetic acid-urea polyacrylamide electrophoresis method to evaluate bovine sperm protamines.

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.

Bone morphogenetic protein 15 supplementation enhances cumulus expansion, nuclear maturation and progesterone production of in vitro-matured bovine cumulus-oocyte complexes.

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.

Influence of seminal plasma during different stages of bovine sperm cryopreservation.

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.

Endoplasmic reticulum distribution during bovine oocyte activation is regulated by protein kinase C via actin filaments.

Fertilization-induced [Ca2+ ]i oscillations generally depend on the release of calcium ions from the endoplasmic reticulum (ER). Since ER is the main store of calcium ions, it plays an important role in oocyte fertilization. However, the mechanism of ER organization at oocyte activation is unknown. Here, we show that protein kinase C (PKC) is involved in ER distribution during bovine oocyte activation, but not involved in cell cycle resumption and spindle organization. Actin filaments were affected by PKC pharmacological inhibition. In addition, similar to PKC results, the actin-depolymerizing drug cytochalasin B affected the ER distribution during oocyte activation. Specifically, we have demonstrated that ER organization during bovine oocyte activation is regulated by PKC possibly through its action on actin filaments regulation. Taken together, the results presented here provide further information on the pathway involved in the regulation of ER organization during oocyte activation and new insight into the functional role of PKC and actin filaments during this process.

Sperm DNA fragmentation: causes and identification.

Sperm DNA fragmentation is referred to as one of the main causes of male infertility. Failures in the protamination process, apoptosis and action of reactive oxygen species (ROS) are considered the most important causes of DNA fragmentation. Action of ROS or changes in sperm protamination would increase the susceptibility of sperm DNA to fragmentation. Routine semen analysis is unable to estimate sperm chromatin damage. Sperm DNA integrity influences sperm functional capability, therefore tests that measure sperm DNA fragmentation are important to assess fertility disorders. Actually, there is a considerable number of methods for assessing sperm DNA fragmentation and chromatin integrity, sperm chromatin stability assay (SCSA modified), sperm chromatin dispersion (SCD), comet assay, transferase dUTP nick end labelling (TUNEL); and protamine evaluation in sperm chromatin assay, such as toluidine blue, CMA3, protamine expression and evaluation of cysteine radicals. This review aims to describe the main causes of sperm DNA fragmentation and the tests commonly used to evaluate sperm DNA fragmentation.

Changes in fertilization medium viscosity using hyaluronic acid impact bull sperm motility and acrosome status.

The female reproductive tract, in particular the composition of the uterine and oviduct fluids, is responsible, at least in part, for triggering sperm cell modifications, essential for the acquisition of fertilization ability. Hyaluronic acid (HA) is a glycosaminoglycan present in these fluids, and its role in the fertilization process and sperm functionality is still barely understood. This work was designed to (a) determine the rheological characteristics of the fertilization medium by the addition of HA and (b) determine the HA influence on sperm motility and functional status. To that end, the in vitro fertilization medium was supplemented with 4 doses of HA (6, 60, 600 and 6,000 µg/ml) and analysed for viscosity and adhesion strength characteristics. Then, thawed semen from 6 bulls were incubated in these media and assessed at 4 different moments for morphological and functional parameters (plasma and acrosomal membrane integrities, mitochondrial membrane potential, capacitation, acrosomal reaction, and motility). The rheological evaluation showed that the addition of HA was able to increase both the viscosity and the adhesion strength of the fertilization medium, especially in the 6,000 µg/ml group in which the effect was more pronounced. No influence of HA could be observed on mitochondrial potential, and acrosomal and plasma membrane integrities. However, HA supplementation, at lower doses, led to an increase in the number of reacted sperm, as well as changes in motility parameters, with increase in the number of motile, rapid and progressive spermatozoa. In conclusion, the addition of HA alters the rheological properties of the fertilization medium and leads to the improvement of the properties related to sperm motility and capacitation, without compromising other functional aspects of the cell.

Immunolocalization of antioxidant enzymes in testis of rams submitted to long-term heat stress.

Long-term heat stress (HS) induced by testicular insulation generates oxidative stress (OS) on the testicular environment; consequently activating antioxidant enzymes such as superoxide dismutase (SOD), glutathione reductase (GR) and glutathione peroxidase (GPx). The aim of this work was to immunolocalize antioxidant enzymes present in different cells within the seminiferous tubule when rams were submitted to HS. Rams were divided into control (n = 6) and treated group (n = 6), comprising rams subjected to testicular insulation for 240 h. After the testicular insulation period, rams were subjected to orchiectomy. Testicular fragments were submitted to immunohistochemistry for staining against SOD, GR and GPx enzymes. We observed immunolocalization of GPx in more cell types of the testis after HS and when compared with other enzymes. In conclusion, GPx is the main antioxidant enzyme identified in testicular cells in an attempt to maintain oxidative balance when HS occurs.

System for evaluation of oxidative stress on in-vitro-produced bovine embryos.

This study proposed a quantitative evaluation of oxidative status (OS) in bovine embryos. Sixteen-cell stage embryos, cultured under 5% O2, were treated with oxidative stress inducer menadione (0, 1, 2.5 and 5 µmol/l) for 24 h. Blastocyst rate (BLR) was recorded and expanded blastocysts were stained with CellROX®Green (CRG; OS evaluation) and evaluated under epifluorescence microscopy (ratio of pixel/blastomere). A significant effect of menadione was observed for BLR (P = 0.0039), number of blastomeres/embryo (P < 0.0001) and OS (P < 0.001). Strong negative correlations were found between BLR and the number of blastomeres with OS evaluation, demonstrating the efficacy of this analysis to evaluate OS levels of IVF bovine embryos.

Cryopreservation of spermatozoa from black-and-gold howler monkeys (Alouatta caraya) using egg yolk-based or soy lecithin-based extenders.

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.

Can in vitro embryo production be estimated from semen variables in Senepol breed by using artificial intelligence?

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.

Effects of different cryopreservation methods on post-thaw culture conditions of in vitro produced bovine embryos.

The aim of this work was to evaluate the effect of cryopreservation protocols on subsequent development of in vitro produced bovine embryos under different culture conditions. Expanded in vitro produced blastocysts (n = 600) harvested on days 7-9 were submitted to controlled freezing [slow freezing group: 10% ethylene glycol (EG) for 10 min and 1.2°C/min cryopreservation]; quick-freezing [rapid freezing group: 10% EG for 10 min, 20% EG + 20% glycerol (Gly) for 30 s]; or vitrification [vitrification group: 10% EG for 10 min, 25% EG + 25% Gly for 30 s] protocols. Control group embryos were not exposed to cryoprotectant or cryopreservation protocols and the hatching rate was evaluated on day 12 post-insemination. In order to evaluate development, frozen-thawed embryos were subjected to granulosa cell co-culture in TCM199 or SOFaa for 4 days. Data were analyzed by PROC MIXED model using SAS Systems for Windows®. Values were significant at p < 0.05. The hatching rate of the control group was 46.09%. In embryos cultured in TCM199, slow freezing and vitrification group hatching rates were 44.65 ± 5.94% and 9.43 ± 6.77%, respectively. In embryos cultured in SOFaa, slow freezing and vitrification groups showed hatching rates of 11.65 ± 3.37 and 8.67 ± 4.47%, respectively. In contrast, the rapid freezing group embryos did not hatch, regardless of culture medium. The slow freezing group showed higher hatching rates than other cryopreservation groups. Under such conditions, controlled freezing (1.2°C/min) can be an alternative to cryopreservation of in vitro produced bovine embryos.

What is known so far about bull sperm protamination: a review.

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.

Paternal age impairs in vitro embryo and in vivo fetal development in murine.

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.

Effect of culture media on porcine embryos produced by in vitro fertilization or parthenogenetic activation after oocyte maturation with cycloheximide.

This study evaluated the effects of reversible meiotic inhibition and different culture media (PZM3 or NCSU23) on production of porcine embryos by either in vitro fertilization (IVF) or parthenogenetic activation (PA). Oocytes from abattoir-derived ovaries were allocated into two groups for maturation: CHX (5 µg/ml cycloheximide for 10 h) or Control (no CHX). The percentage of metaphase II (MII) oocytes was determined at 36, 40 or 44 h of in vitro maturation. For IVF and PA, denuded oocytes were fertilized with purified sperm for 6 h or activated by electric stimuli. Zygotes were then subdivided into two culture groups: NCSU23 or PZM3. No effect of treatment with CHX and culture media was observed on cleavage (D3) and blastocyst (D7) rates in IVF and PA groups. There are no differences of quality or development rates between IVF-derived embryos cultured in NCSU23 or PZM3. However, we observed high quality PA embryos in PZM3 compared with NCSU23. Maturation arrest with CHX decreased the average blastocyst cell number in IVF while it was increased in PA embryos. As older oocytes are more effectively activated, CHX- blocked oocytes reached the mature stage faster than the control group. In conclusion, the CHX treatment for 10 h, followed by oocyte maturation for 40 h, is an efficient protocol to produce high quality parthenote embryos, especially when they are cultured in PZM3. However, this protocol is not satisfactory for IVF embryos production. In this case, a shorter maturation period could provide better embryo quality.

Myostatin gene knockdown through lentiviral-mediated delivery of shRNA for in vitro production of transgenic bovine embryos.

Myostatin is described as a negative regulator of the skeletal muscle growth. Genetic engineering, in order to produce animals with double the muscle mass and that can transmit the characteristic to future progeny, may be useful. In this context, the present study aimed to analyse the feasibility of lentiviral-mediated delivery of short hairpin RNA (shRNA) targeting of myostatin into in vitro produced transgenic bovine embryos. Lentiviral vectors were used to deliver a transgene that expressed green fluorescent protein (GFP) and an shRNA that targeted myostatin. Vector efficiency was verified through in vitro murine myoblast (C2C12) cell morphology after inductive differentiation and by means of real-time PCR. The lentiviral vector was microinjected into the perivitellinic space of in vitro matured oocytes. Non-microinjected oocytes were used as the control. After injection, oocytes were fertilized and cultured in vitro. Blastocysts were evaluated by epifluorescence microscopy. Results demonstrated that the vector was able to inhibit myostatin mRNA in C2C12 cells, as the transducted group had a less amount of myostatin mRNA after 72 h of differentiation (p < 0.05) and had less myotube formation than the non-transduced group (p < 0.05). There was no difference in cleavage and blastocyst rates between the microinjected and control groups. After hatching, 3.07% of the embryos exhibited GFP expression, indicating that they expressed shRNA targeting myostatin. In conclusion, we demonstrate that a lentiviral vector effectively performed shRNA myostatin gene knockdown and gene delivery into in vitro produced bovine embryos. Thus, this technique can be considered a novel option for the production of transgenic embryos and double muscle mass animals.

Bovine sperm cells viability during incubation with or without exogenous DNA.

SummaryThe aim of this study was to assess the effect of exogenous DNA and incubation time on the viability of bovine sperm. Sperm were incubated at a concentration of 5 x 106/ml with or without plasmid pEYFP-NUC. Fluorescent probes, propidium iodide/Hoechst 33342, FITC-PSA and JC-1, were used to assess plasma membrane integrity (PMI), acrosome membrane integrity (AMI) and mitochondrial membrane potential (MMP) respectively at 0, 1, 2, 3 and 4 h of incubation. Exogenous DNA addition did not affect sperm viability; however, incubation time was related to sperm deterioration. Simultaneous assessment of PMI, AMI and MMP showed a reduction in the number of sperm with higher viability (integrity of plasma and acrosome membranes and high mitochondrial membrane potential) from 58.7% at 0 h to 7.5% after 4 h of incubation. Lower viability sperm (damaged plasma and acrosome membranes and low mitochondrial membrane potential) increased from 4.6% at 0 h to 25.9% after 4 h of incubation. When PMI, AMI and MMP were assessed separately we noticed a reduction in plasma and acrosome membrane integrity and mitochondrial membrane potential throughout the incubation period. Therefore, exogenous DNA addition does not affect sperm viability, but the viability is reduced by incubation time.

Sperm traits on in vitro production (IVP) of bovine embryos: Too much of anything is good for nothing.

Sperm samples used on fertilization strongly influence the in vitro production (IVP) rates. However, sperm traits behind this effect are not stated consistently until now. This study aimed to evaluate the isolated and combined effect of some sperm traits (MB: total motility before Percoll® gradient, MA: total motility after Percoll® gradient, AI: acrosome integrity, MI: membrane integrity, MP: mitochondrial membrane potential, and CR: chromatin resistance) on IVP rates. This is the first study focusing on the isolated effect of distinct traits. For this purpose, the experiment was divided in three steps. In first step, to study behavior of traits sperm samples (n = 63 batches) were analyzed and ranked based on each trait. In second step, samples ranked were selected from target ranks regions and allocated in groups of four to five batches, creating Higher and Lower groups, according to two different approaches. One aimed to form groups that differed to all sperm traits simultaneously (effect of combined traits). The other aimed to form groups that differed only to a single sperm trait while no differences were observed for the remaining traits (effect of each isolated trait). In third step, for each group successfully formed in step 2, sperm samples were individually and prospectively used for IVP. Cleavage, embryo development and blastocyst rates were recorded and compared between Higher and Lower of respective trait groups. Surprisingly, evaluation of isolated effects revealed that lower levels of MB, AI and MP resulted in higher embryo development and blastocyst rates (p<0.05), which was not observed on cleavage rate. We conclude that sperm traits strongly influence embryo development after in vitro fertilization (IVF), affecting the zygote competence to achieve blastocyst stage. Individually, levels of MB, AI or MP could be some of the key traits that may define IVP efficiency on current systems of embryo production.

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.

Effect of Heat Stress on Sperm DNA: Protamine Assessment in Ram Spermatozoa and Testicle.

Sperm DNA fragmentation is considered one of the main causes of male infertility. The most accepted causes of sperm DNA damage are deleterious actions of reactive oxygen species (ROS), defects in protamination, and apoptosis. Ram sperm are highly prone to those damages due to the high susceptibility to ROS and to oxidative stress caused by heat stress. We aimed to evaluate the effects of heat stress on the chromatin of ejaculated and epididymal sperm and the activation of apoptotic pathways in different cell types in ram testis. We observed higher percentages of ejaculated sperm with increased chromatin fragmentation in the heat stress group; a fact that was unexpectedly not observed in epididymal sperm. Heat stress group presented a higher percentage of spermatozoa with DNA fragmentation and increased number of mRNA copies of transitional protein 1. Epididymal sperm presented greater gene expression of protamine 1 on the 30th day of the spermatic cycle; however, no differences in protamine protein levels were observed in ejaculated sperm and testis. Localization of proapoptotic protein BAX or BCL2 in testis was not different. In conclusion, testicular heat stress increases ram sperm DNA fragmentation without changes in protamination and apoptotic patterns.