C. Cheuquemán

Sperm Membrane Functionality in the Dog Assessed by Flow Cytometry

The objective assessment of sperm function increases the chances of predicting the fertilizing capacity of a fresh semen sample or diagnosing infertility problems. In this study, the available flow cytometry technique was used to determine the membrane functional capacity of canine spermatozoa. The second fractions of ejaculates from six dogs were pooled, and samples (n = 26) processed to determine the variables: sperm viability and plasma membrane integrity by Sybr-14/Pi staining; phosphatidylserine (PS) translocation by Annexin-V-FITC/PI labelling; acrosome membrane integrity by FITC-conjugated Pisum sativum agglutinin/PI labelling; and mitochondrial membrane potential (ΔΨm) by staining with JC-1. Means for the 26 examined samples indicated that 82.66 ± 2.8% of the viable spermatozoa showed an intact plasma membrane, 8.4 ± 2.6% were moribund, 72.7 ± 16% had an intact acrosome, 80.9 ± 17% had high ΔΨm and 8.1 ± 11% had PS translocation with a PS translocation index of 2.1 ± 3%. Motility was only correlated with PS translocation (R = 0.3901; p = 0.0488), and acrosome membrane integrity was correlated with PS translocation (R = -0.5816; p = 0.0018). This study provides objective physiological data on the functional capacity of canine spermatozoa.

Supplementation of IVF medium with melatonin: effect on sperm functionality and in vitro produced bovine embryos

Gamete co‐incubation generates high free radical levels surrounding growing zygotes which may impair subsequent embryo viability. Melatonin eliminates a wide variety of free radicals; hence, we tried to improve in vitro embryo production by adding melatonin to in vitro fertilisation (IVF) media in high (Exp. 1) and low concentrations (Exp. 2), and we evaluated its effect on bull sperm function during IVF co‐incubation time (Exp. 3). In Experiment 1, we supplemented IVF media culture with 0.01, 0.1 and 1 mmol of melatonin, along with a no melatonin control group. In Experiment 2, melatonin levels were reduced to 10, 100 and 1000 nmol, with a no melatonin control group. In Experiment 3, spermatozoa were incubated in IVF media with melatonin (as Exp. 2) and functional parameters were analysed at 0, 4 and 18 h. In Experiment 1, only 1 mmol melatonin showed lesser blastocyst rates than control (C: 23.2 ± 6.7% versus 1 mmol: 2.0 ± 1.7%). In Experiment 2, no statistical differences were found in cleavage percentage, blastocyst percentage and total cell count for any melatonin treatment. In Experiment 3, sperm samples with 1000 nmol melatonin had a significantly higher wobbler (WOB) coefficient, a lower percentage of intact acrosomes, a lower percentage of viable spermatozoa with ROS, greater DNA fragmentation and higher DNA oxidation than controls. Total fluorescence intensity for ROS at 10 nmol melatonin was significantly greater than controls (P < 0.05). IVF media with 1 mmol melatonin is deleterious for embryo development, and in lower concentrations, it modulated sperm functionality, but had no effects on embryo production.

Effects of short-term exposure of mature oocytes to sodium nitroprusside on in vitro embryo production and gene expression in bovine.

Stress tolerance can be induced in embryos by oocyte exposure to hydrostatic pressure, osmotic agents, heat shock, or reactive oxygen species. This study assessed the effects of exposing bovine oocytes to a nitric oxide (NO) donor, sodium nitroprusside (SNP), on subsequent in vitro embryo production, embryo quality and the expression of genes involved in NO production (iNOS, eNOS, and nNOS), stress tolerance (HSP70 and HSP90), oxidative stress (HIF1A and PRDX5), and apoptosis (BCL2A1). In vitro mature oocytes were incubated with SNP (control, 10(-6) M, 10(-5) M, and 10(-4) M) for 1 hour before in vitro fertilization, and cultured until Day 7. Cleavage and blastocyst rates were recorded. Next, embryo quality (ratio of inner cell mass to total cell number) and relative gene expression of iNOS, eNOS, nNOS, HSP70, HSP90, HIF1A, PRDX5, and BCL2A1 were determined in expanded blastocysts. Cleavage rates were significantly lower for 10(-4) M SNP compared with the control and 10(-5) M SNP treatments (77 ± 7.1%, 82 ± 8.4%, and 84.9 ± 4.1%, respectively). Total blastocyst rates were lower in the 10(-4) M SNP group relative to the control group (26.2 ± 4.9% and 34.1 ± 7.8%, respectively). Embryo quality was similar among the groups. However, our relative gene expression analysis revealed the downregulation of endothelial oxide nitric synthase messenger RNA in expanded blastocysts in all the treatment groups compared with the control treatment. These results suggest that the short-term exposure of mature bovine oocytes to a NO donor does not induce their stress tolerance and has no beneficial effect on the in vitro embryo production of bovine embryos.

Melatonin Scavenger Properties against Oxidative and Nitrosative Stress: Impact on Gamete Handling and In Vitro Embryo Production in Humans and Other Mammals

Oxidative and nitrosative stress are common problems when handling gametes in vitro. In vitro development in mammalian embryos is highly affected by culture conditions, especially by reactive oxygen species (ROS) and reactive nitrogen species (RNS), because their absence or overproduction causes embryo arrest and changes in gene expression. Melatonin in gamete co-incubation during in vitro fertilization (IVF) has deleterious or positive effects, depending on the concentration used in the culture medium, demonstrating the delicate balance between antioxidant and pro-oxidant activity. Further research is needed to better understand the possible impact of melatonin on the different IVP steps in humans and other mammals, especially in seasonal breeds where this neuro-hormone system highly regulates its reproduction physiology.

Short-term storage of salmonids semen in a sodium alginate-based extender.

Short‐term storage of semen is a useful strategy for preservation of fish spermatozoa. However, there is a significantly decrease on sperm function mainly due to oxidative stress. In this way, sodium alginate plays an important role as free radical scavenger compound. Accordingly, the aim of our study was to analyse the effect of a sodium alginate‐based extender on sperm function in the short‐term storage of salmonids semen. Samples of Salmo salar, Oncorhynchus kisutch, and Oncorhynchus mykiss were stored in Storfish® (Ext‐C) and Storfish® supplemented with sodium alginate (Ext‐A) during 10 days at 4°C. After storage, motility, viability, mitochondrial membrane potential (ΔΨmit), superoxide anion (O2−) level and DNA fragmentation (DNA Frag) were assessed. Ext‐A had positive effect in preservation of sperm motility, viability, ΔΨmit, O2− level and DNA integrity in the three species analysed compared to control samples. In Ext‐A, the spermatozoa of S. salar and O. mykiss showed significantly higher motility, viability and ΔΨmit than O. kisutch. However, O. kisutch and O. mykiss had significantly lower O2− level than S. salar, and DNA fragmentation in O. kisutch and S. salar was significantly lower than in samples of O. mykiss (p < 0.05). Dilution of salmonids semen in a sodium alginate‐based extender is effective for protecting sperm quality during 10 days of short‐term storage.

Effect of the age of broodstock males on sperm function during cold storage in the trout (Oncorhynchus mykiss)

The knowledge of sperm quality in the broodstock males of different ages is a prerequisite to identify the reproductive ability of cultivated fish for the hatchery management. Thus, in this work, we analysed sperm function of the semen stored of broodstock males of rainbow trout (Oncorhychus mykiss) in different reproductive ages (2, 3 and 4 years old). Sperm samples of each reproductive age were stored in Storfish® during 10 days at 4°C, and then, motility, viability, mitochondrial function (MMP), superoxide anion ( O2− ) level and DNA fragmentation (DNAfrag) were assessed. The results demonstrated that sperm function parameters were affected significantly by the age of the males and the time of storage. Motility, viability and MMP significantly decreased, and DNAfrag and O2− level increased with the age increment and the time of storage. In conclusion, sperm quality of 2 and 3 years old were superior to those of 4 years old, based on higher quality of various sperm functions such as motility, viability, MMP, DNA integrity and level O2− during short‐term storage. This information must be considered for optimum utilization of broodstock males in aquaculture.

Decrease in bovine in vitro embryo production efficiency during winter season in a warm‐summer Mediterranean climate

Retrospective analysis of monthly embryo production from December 2011 to May 2015 and its correlation with meteorological data in our geographic zone was made. We had observed that in certain time of the year, in vitro blastocyst production decreases. Accordingly, was examined the association between blastocyst production and climatological parameters. Cleavage rates correlate positively with blastocyst rates (p < .05). Significant differences in cleavage rates between autumn and summer (79.8%; 71.5%), and between winter and autumn (71.8%; 79.8%), were found. Blastocyst production had lower efficiency in June (9 ± 12%) and July (4.9 ± 5.7%), which coincides with winter season. In contrast, higher embryo production was obtained in February (22.2 ± 9.7%), March (22.9 ± 14%) and September (25.2 ± 6.6%), which coincides with autumn and spring season. Similarly, embryo production correlates with meteorological parameters: blastocyst production positively correlates with sunshine hours, maximum temperature and average temperature. Similarly, blastocyst production inversely correlates with total precipitation and days >1 mm precipitation (p < .05). There is a significant decrease in bovine in vitro embryo production efficiency during winter season in our warm‐summer Mediterranean climate zone. It remains to be investigated the direct effect of environmental factors on oocyte quality and its impact on in vitro production efficiency.

Changes in sperm function and structure after freezing in domestic cat spermatozoa

Sperm cryopreservation allows for a long‐term storage of genetic. However, changes due to factors as cold shock, osmotic and oxidative stress cause reduction in viability and fertilising ability of frozen/thawed spermatozoa. Therefore, evaluation of cryoinjury of cat spermatozoa is a key factor in achieving better cryopreservation results. This study analysed the changes in structural and functional after freezing in ejaculated domestic cats spermatozoa. Semen samples (n = 60) were analysed before and after freezing, progressive motility was determined with computer‐assisted sperm analysis and viability, and acrosome intact spermatozoa, mitochondrial function and superoxide anion ( O2- ) were assessed by flow cytometry. The results demonstrated that cryopreservation induced changes in all sperm parameters (p < 0.05). Total sperm motility, viability, acrosome integrity and mitochondrial function of fresh samples were near to 80% and decrease near to 40% in frozen/thawed spermatozoa (p < 0.05); nevertheless, in contrast to all other sperm parameters, the sperm positive with O2- increased post/thawing (p < 0.05). In conclusion, changes in frozen/thawed spermatozoa could be related to the effect of oxidative stress due to the increase in the synthesis of O2- and a concomitant loss of functional competence. Therefore, the evaluation of these sperm parameters could contribute to complement the analysis of fresh or frozen semen used ART.

Effect of Sperm Selection Techniques in Frozen/Thawed Cat Spermatozoa on Sperm Motility Analyzed by CASA System

Freeze/thawing process reduces sperm survival and fertilizing ability of cat spermatozoa, with sperm motility being the most sensitive sperm parameter altered, due to cryo-damage. In this context, swim-up and density gradient processing method can help to recover high motile and normal spermatozoa. Maximizing the use of frozen semen sample is essential, especially in e ndangered felids or high value cats in which sample size, number of samples or access to semen collection is reduced. To our knowledge, t here is no previous report describing an in depth analysis of sperm motility improvement, after sperm selection techniques in frozen cat s em . Accordingly, we evaluated the effect of percoll gradient (PG) and swim up (SU) sperm selection techniques on sperm motility par ameters and sperm recovery rate in frozen/thawed spermatozoa of domestic cat. Next, we evaluated the individual effect of the cat over sperm motility after PG sperm selection of frozen/thawed spermatozoa. SU and PG improved significantly all sperm motility parameters of frozen/thawed cat spermatozoa compared to simple washing. However, PG allows better sperm recovery from the original frozen sam ple and works mostly homogeneously among individual cats. This new information could help to maximize the use of frozen semen in endangered felids or high value domestic cats for its subsequent application on in vitro fertilization and artificial insemination.