Establishing the normal range of sperm DNA fragmentation index (% DFI) for rhesus macaques.

The Sperm Chromatin Structure Assay (SCSA) is a robust test with high repeatability and precision. It is a clinically accepted assay that defines risk for infertility in men by measuring the degree of DNA fragmentation (% DFI) in sperm. The objective of this study was to adapt and validate the SCSA for rhesus macaques (Macaca mulatta) and establish a range for % DFI in fertile males. Sperm samples from two different males were used to produce a % DFI validation curve before establishing a range using additional samples from n = 11 males. Sperm labeled with acridine orange were analyzed by flow cytometry to measure green fluorescence (native or intact DNA) and red fluorescence (fragmented DNA). Data were exported to FlowJo software to determine the % DFI for each sample. DNA fragmentation ranged from 0.1 to 2.4% DFI, with a mean ± SD = 1.1 ± 0.7% DFI (validation curve optimized to R2 > 0.95). In conclusion, we were able to successfully validate the SCSA in our institution and establish the first normal range for sperm DNA fragmentation in rhesus macaques. Our study provides a quantitative baseline for future evaluations to assess macaque fertility through the SCSA test.

Spermatogonial stem cell potential of CXCR4-positive cells from prepubertal bull testes.

Spermatogonial stem cells (SSC) have the potential to restore spermatogenesis when transplanted into testes depleted of germ cells. Due to this property, SSC could be used in breeding programs and in transgenic animal research. Particularly in cattle, SSC are not as well characterized as in mice or humans. In mice, C-X-C Motif Chemokine Receptor 4 positive (CXCR4+) testicular cells have high SSC potential. It, therefore, was hypothesized that CXCR4 is a marker of undifferentiated spermatogonia in cattle. Using samples from pre-pubertal calves, the CXCR4 protein was detected by immunohistochemistry in a few cells of the seminiferous tubules. Testicular cells were isolated, frozen-thawed and submitted to magnetic-activated cell sorting using anti-CXCR4 antibody. Quantitative RT-PCR analysis revealed that CXCR4+ cells had THY1, OCT4 and ZBTB16 (or PLZF) mRNA in these cells. Flow cytometry results indicated that the proportion of THY1+ cells is enriched in CXCR4+ populations. Colonization potential of CXCR4+ cells was assessed after xenotransplantation into testes of nude mice treated with busulfan. Transplantation of CXCR4+ cells yielded an increase of 5.4-fold when compared to CXCR4- cells. These results indicate that CXCR4 could be used as a marker to enrich and sort cells of bulls with putative spermatogonial stem cell potential.

The addition of docosahexaenoic acid (DHA) and antioxidants (glutathione peroxidase and superoxide dismutase) in extenders to epididymal sperm cryopreservation in bulls.

SummaryThe cryopreservation of epididymal sperm is an important technique that allows genetic material to be preserved, even post mortem. However, cryopreservation leads to increased oxidative stress and impaired sperm viability. Polyunsaturated fatty acid (PUFA) supplementation may improve certain sperm characteristics, but it also makes sperm more susceptible to oxidative stress, therefore adding antioxidants that counteract oxidative stress has become an option. In this context, this study aimed to evaluate the effect of the interaction between docosahexaenoic acid (DHA) and antioxidants on the quality after the cryopreservation of epididymal bull sperm. Twenty epididymides were collected after slaughter, and epididymal sperm was cryopreserved with bovine extender supplemented with docosahexaenoic acid (DHA), glutathione peroxidase (GPx) and superoxide dismutase (SOD). We verified an improvement in motility in the group that was treated only with DHA 5 µM and a concentration-dependent effect on susceptibility to lipid peroxidation that was associated with DHA concentration (1 µM, 5 µM or 10 µM). Moreover, treatment with DHA (5 µM) and SOD (20 IU/ml) resulted in higher sperm motility. Thus, the association between DHA (5 µM) and SOD (20 IU/ml) appears to be an option for increased epididymal sperm features in bulls.

Effects of Soy Lecithin Extender on Dog Sperm Cryopreservation.

Semen cryopreservation is an essential biotechnology in canine reproduction and during the cryopreservation process commonly egg yolk are used. The discrepancy in the egg yolk composition and the potential risk of disease dissemination are obstacles for semen exportation and use. Therefore, studies aiming to substitute egg yolk are extremely important. In this context, soy lecithin contains a low-density lipoprotein fraction, is an interesting alternative. Thus, the objective of this study was to compare extenders based on soy lecithin (several concentrations and forms) with egg yolk during the cryopreservation process of dog sperm. For this purpose, we used twelve dogs. Semen was evaluated at different time points (after refrigeration, glycerolization, and thawing), by motility analysis (CASA) and functional tests (e.g., membrane integrity-eosin/nigrosin, acrosome integrity-fast green/Bengal rose, mitochondrial activity-3'3 diaminobenzidine, Chromatin susceptibility to acid-induced denaturation-SCSA, and susceptibility to oxidative stress-thiobarbituric acid reactive substances). The results indicated that egg yolk and lower concentrations of lecithin had similar effects on mitochondrial activity and motility. Thus, soy lecithin is a potentially viable alternative to egg yolk for the cryopreservation of dog semen.

Effect of Vitamin E and Polyunsaturated Fatty Acids on Cryopreserved Sperm Quality in Bos taurus Bulls Under Testicular Heat Stress.

Taurine bulls are highly susceptible to heat stress, leading to increased oxidative stress (OS) and impaired sperm viability. Polyunsaturated fatty acids (PUFAs) supplementation can be an alternative to improve semen quality, which also results in more sperm susceptibility to lipid peroxidation. Moreover, this deleterious effect can be exacerbated in animals affected by heat stress. Vitamin E is a key antioxidant that counteracts lipid peroxidation of sperm membrane caused by OS. Thus, combining PUFAs with vitamin E may improve sperm quality. In this context, this study aimed to evaluate the effect of interaction between PUFAs and vitamin E on sperm quality in Bos taurus bulls under testicular heat stress. Sixteen taurine bulls under testicular heat stress were randomly assigned in four groups: Control, Vitamin E, PUFA, and PUFA + Vitamin E. All groups lasted for 60 days. Samples were cryopreserved/thawed and analyzed for motility variables (CASA), membrane and acrosome integrity, mitochondrial activity, susceptibility to oxidative stress, DNA integrity, and sperm-binding capacity. Results showed that vitamin E had a beneficial effect on some sperm characteristics, whereas PUFA supplementation had an adverse effect when the two treatments were evaluated separately. Finally, the association between PUFAs and vitamin E did not improve sperm quality.

Effects of photobiomodulation therapy (PBMT) on bovine sperm function.

Fertilization rates and subsequent embryo development rely on sperm factors related to semen quality and viability. Photobiomodulation therapy (PBMT) is based on emission of electromagnetic waves of a laser optical system that interact with cells and tissues resulting in biological effects. This interaction is mediated by photoacceptors that absorb the electromagnetic energy. Effects are dependent of irradiation parameters, target cell type, and species. In sperm, PBMT improves several features like motility and viability, affecting sperm aerobic metabolism and energy production. The aim of this study was to investigate, under same conditions, how different output powers (5, 7.5, and 10 mW) and time of irradiation (5 and 10 min) of laser (He-Ne laser, 633 nm) may affect frozen/thawed bovine sperm functions. Results showed significant effects depending on power while using 10 min of irradiation on motility parameters and mitochondrial potential. However, no effect was observed using 5 min of irradiation, regardless of power applied. In conclusion, PBMT is effective to modulate bovine sperm function. The effectiveness is dependent on the interaction between power applied and duration of irradiation, showing that these two parameters simultaneously influence sperm function. In this context, when using the same fluency and energy with different combinations of power and time of exposure, we observed distinct effects, revealing that biological effects should be also based on simple parameters rather than only composite parameters such as fluency, irradiance and energy. Laser irradiation of frozen/thawed bovine semen led to an increase on mitochondrial function and motility parameters that could potentially improve fertility rates.

Sperm Oxidative Stress Is Detrimental to Embryo Development: A Dose-Dependent Study Model and a New and More Sensitive Oxidative Status Evaluation.

Our study aimed to assess the impact of sperm oxidative stress on embryo development by means of a dose-dependent model. In experiment 1, straws from five bulls were subjected to incubation with increasing H2O2 doses (0, 12.5, 25, and 50 µM). Motility parameters were evaluated by Computed Assisted System Analysis (CASA). Experiment 2 was designed to study a high (50 µM) and low dose (12.5 µM) of H2O2 compared to a control (0 µM). Samples were incubated and further used for in vitro fertilization. Analyses of motility (CASA), oxidative status (CellROX green and 2'-7' dichlorofluorescein diacetate), mitochondrial potential (JC-1), chromatin integrity (AO), and sperm capacitation status (chlortetracycline) were performed. Embryos were evaluated based on fast cleavage (30 h.p.i.), cleavage (D = 3), development (D = 5), and blastocyst rates (D = 8). We observed a dose-dependent deleterious effect of H2O2 on motility and increase on the percentages of positive cells for CellROX green, capacitated sperm, and AO. A decrease on cleavage and blastocyst rates was observed as H2O2 increased. Also, we detected a blockage on embryo development. We concluded that sperm when exposed to oxidative environment presents impaired motility traits, prooxidative status, and premature capacitation; such alterations resulting in embryo development fail.