Induction of oxidative stress does not increase the cryotolerance of vitrified embryos.

Short-term treatment of mammalian oocytes with different stressors induces stress tolerance of embryos derived from these oocytes. The aims of this study were to evaluate effects on embryo development when there was treatment of oocyte complexes (COCs) used to derive the embryos with hydrogen peroxide (H2O2).The COCs were not incubated with H2O2: control (0 µM), or were incubated with 25, 50, 75, or 100 µM concentrations of H2O2 for 1 h prior to in vitro fertilization, and presumptive zygotes were cultured until day 7. Blastocysts at day 7 of development derived from H2O2-treated (25 µM treatment concentration) COCs were vitrified. Percentage of embryos undergoing cleavage was not affected by any treatment, while percentage of embryos developing to the blastocyst stage was less when there was treatment of COCs with 100 µM of H2O2. Embryo quality was less when COCs used to derive blastocysts were treated with 50, 75, or 100 µM concentrations of H2O2. There were lesser relative abundances of some mRNA transcripts of interest in blastocysts when there was treatment of COCs with H2O2. After vitrification, there were no differences in embryo re-expansion and hatching rates compared with fresh and vitrified blastocysts of the control group and those derived from COCs treated with 25 µM H2O2. In conclusion, treatment of COCs used to derive blastocysts with H2O2 does not induce stress tolerance in vitrified embryos of cattle; however, the viability of these blastocysts is similar to those of the control group.

Effect of high-fat and vitamin D deficient diet on rat sperm quality and fertility.

Vitamin D (VD) is involved in many functions of the reproductive system male. The intake of diets high-fat and vitamin D deficiency (VD-) can cause morphological and physiological changes in testis that relate to infertility in the male. However, its effects on sperm quality and in vivo fertility have been little studied. This study analyzed the effects of fat and VD on sperm quality and in vivo fertility in sperm of Sprague-Dawley rats. The rats were divided into four groups: G1: control diet with vitamin D (C/VD+), G2: control diet without vitamin D (C/VD-), G3: high-fat diet with vitamin D (HF/VD+) and G4: high-fat diet without vitamin D (HF/VD-) and were fed for 3 months. Adipose tissue weight and plasma glucose were determined. Sperm quality was analyzed for motility (MO), mitochondrial function and fertilizing capacity. The intake of a high-fat diet caused a significant increase in body weight of rats (P < 0.05). There were fat-by-VD interaction effects (P < 0.05) on MO and MMP. MO and MMP were greater (P < 0.05) in G1 (54 ±â€¯5.5% and 60.5 ±â€¯2.6%) than in G3 and G4 (20 ±â€¯6.0% and 27.7 ±â€¯3.6), whereas G2 (36.7 ±â€¯8.9% and 30.7 ±â€¯4.2%) was intermediate. There was no fat-by-VD interaction for fertilizing capacity. However, fertilizing capacity was greater (P > 0.05) in animals receiving control diet (70 ±â€¯21%); than in animals receiving high-fat diet (20 ±â€¯11%). Our results demonstrated that the high-fat diet and VD- contribute to the decrease in sperm quality (MO, MMP) and consequently could decrease the fertilizing capacity.

Effects of Diet-Induced Obesity and Deficient in Vitamin D on Spermatozoa Function and DNA Integrity in Sprague-Dawley Rats.

Obesity has adverse effects on male fertility and usually is diagnosed with a prevalence of vitamin D deficiency (VD-). Discussion on the impact of obesity/VD- on sperm function has been limited. This study analyzed the effects of diet-induced obesity/VD- on viability and plasma membrane integrity (PMI), superoxide anion (O2 -) level, and DNA fragmentation (DNAfrag) in sperm Sprague-Dawley rats. The males were randomized into four groups and fed for a period of 12 weeks: G1: control diet with vitamin D (C/VD+), G2: control diet without vitamin D (C/VD-), G3: high-fat diet with vitamin D (HF/VD+), and G4: high-fat diet without vitamin D (HF/VD-). Sperm function parameters were analyzed by flow cytometry. PMI percentages and O2 - levels were not affected by any of the diets. DNA fragmentation was increasing significantly (p<0.05) in the spermatozoa of animals with diets vitamin D deficient (G2) and diet-induced obesity (G4). Our results allow us to point out that diet-induced obesity and VD- produce greater damage in DNA sperm of rats. The use of nutraceuticals containing vitamin D could be reducing the risk of fragmentation of DNA in spermatozoa.

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.

Effect of sperm selection techniques in frozen/thawed cat spermatozoa on sperm motility analyzed by CASA system / Efecto de técnicas de selección espermática en espermatozoides congelados/descongelados de gato sobre la motilidad espermática analizada mediante sistema CASA

SUMMARY: 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 methods can help to recover high motile and normal spermatozoa. Maximizing the use of frozen semen sample is essential, especially in endangered felids or high value cats in which sample size, number of samples or access to semen collection is reduced. To our knowledge, there is no previous report describing an in depth analysis of sperm motility improvement, after sperm selection techniques in frozen cat semen. Accordingly, we evaluated the effect of percoll gradient (PG) and swim up (SU) sperm selection techniques on sperm motility parameters 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 sample 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.

RESUMEN: El proceso de congelación/descongelación reduce la sobrevivencia espermática y la habilidad para fertilizar en los espermatozoides de gato, siendo la motilidad espermática el parámetro más sensiblemente alterado debido al daño por frío. En este contexto, los métodos de procesamiento de swim-up y gradiente de densidad pueden ayudar a recuperar los espermatozoides normales y de alta motilidad. Maximizar el uso de una muestra de semen congelado es esencial, especialmente en felinos amenazados o en gatos de alto valor en los cuales el tamaño de muestra, número de muestras o el acceso a la colecta de semen son reducidos. Para nuestro conocimiento, no hay reportes previos que describan un análisis profundo del mejoramiento de la motilidad luego de técnicas de selección espermática en semen congelado de gato. De acuerdo a esto, evaluamos el efecto de las técnicas de selección espermática gradiente de percoll (PG) y swim up (SU) sobre los parámetros de motilidad y porcentaje de recuperación de espermatozoides congelados/descongelados de gato doméstico. Luego, evaluamos el efecto individual del gato sobre la motilidad espermática luego de la selección espermática con PG en espermatozoides congelados/descongelados. SU y PG mejoraron significativamente todos los parámetros de motilidad espermática de los espermatozoides congelados/descongelados comparado con el lavado simple. Sin embargo, PG permitió una mejor recuperación de espermatozoides desde la muestra congelada original y funcionó en su mayoría de manera homogénea entre los gatos individualmente. Esta nueva información puede ayudar a maximizar el uso del semen congelado en felinos amenazados o en gatos de alto valor para su posterior aplicación en fecundación in vitro e inseminación artificial.

Role of vitamin D in the development of obesity / Rol de la vitamina D en el desarrollo de la obesidad

SUMMARY: Antecedents in the literature suggest that vitamin D (VD) play a role in overweigh/obesity. The present study evaluated the effect of VD deficiency diet intake and fat hight on overweight/obesity about white adipose tissue (WAT) and body mass (BM) gain. Animals were divided into four experimental groups according to the lipid and VD content of their diets; G1: CVD+ (C: control diet with VD+; n=5), G2: CVD- (control diet without VD-; n=5), G3: HFVD+ (high fat diet, with VD+; n=5), G4: HFVD- (HF diet without VD-; n=5). The diets were administered for three months and BW was monitored weekly. At the end of this period all animals were euthanized. Epididymal (EFM), retroperitoneal (RFM) and subcutaneous (SFM) fat mass were removed, weighted. At 12 weeks the body mass of the animals that were fed without VD- diets; G2: 507.60±17.31 g, and G4: 528.50±13.50 g were significantly higher (p < 0.05), than the counterparts G1: 485.0±11.29 g and G3: 521.20±26.20 g respectively. Similarly, the animals fed with VDdiets had a greater EFM and SFM (p < 0.05) compared with the respective controls (VD+). Nevertheless, the animals fed with high fat diet had equal RFM (G3: 12.2±4.10 g, G4: 12.88±2.3 g, p > 0.05). The results demonstrate that the nutrition of rats with diet deficient in VD and high fat, promotes overweight by increasing fat deposits, suggestion a cause-effect relationship between VD deficiency and overweight. These results suggest that VD deficiency increases the risk of visceral fat obesity in males.

RESUMEN: Los antecedentes de la literatura sugieren una relación entre la vitamina D (VD) y el sobrepeso/obesidad, sin embargo, causalidad de la relación no está clara. El presente estudio evaluó el efecto de la ingesta dietética deficiente de VD y alta en grasa sobre el tejido adiposo (TA) y la masa corporal (MC). Los animales se dividieron en cuatro grupos experimentales de acuerdo con el contenido de VD y lípido en la dieta; G1: CVD+ (C: dieta control con VD+; n = 5), G2: CVD- (dieta control sin VD-; n = 5), G3: HFVD+ (dieta alta en grasa, con VD+; n = 5), G4: HFVD- (dieta HF sin VD-; n = 5). Las dietas se administraron durante tres meses y MC se controló semanalmente. Al final de este período, los animales fueron sacrificados. La masa grasa epididimaria (MGE), subcutánea abdominal (MGS) y retroperitoneal (MGR) fueron diseccionadas y pesadas individualmente. A las 12 semanas, la MC de los animales alimentados con dietas sin VD-; G2: 507,60 ± 17,31 g, y G4: 528,50 ± 13,50 g fue significativamente mayor (p < 0,05), que sus homólogos G1: 485,0 ± 11,29 g y G3: 521,20 ± 26,20 g respectivamente. De forma similar, los G2 y G4 tuvieron una mayor MGE y MGS (p < 0,05) en comparación con los controles respectivos (VD+). Sin embargo, los animales alimentados con dieta alta en grasas tuvieron igual MGR (G3: 12,2 ± 4,10 g; G4: 12,88 ± 2,3 g, p > 0,05). Los resultados demuestran que la nutrición de ratas con dieta deficiente en VD y alta en grasa, promueve el sobrepeso/obesidad al exacerbar la ganancia de masa grasa en los diferentes depósitos de grasa, sugiriendo una relación causa-efecto entre la deficiencia de VD y el sobrepeso/obesidad. Estos resultados sugieren que la deficiencia de VD aumenta el riesgo de obesidad de grasa visceral en machos.

Freezing dog semen using -80 °C ultra-freezer: Sperm function and in vivo fertility.

Long term storage of canine frozen semen is conventionally performed in liquid nitrogen (LN2). However, previous works in freezing canine semen using a -80 °C ultra-freezer (-80°C-UF) showed no differences on sperm quality after thawing. The main objective of this study was to compare the effects of the freezing techniques using LN2 or -80°C-UF on sperm function and in vivo fertility of frozen-thawed dog semen. The sperm-rich fraction of the ejaculate was collected separately from five Chihuahua breed, and each one divided into two aliquots, and frozen and stored in LN2 or -80°C-UF. Sperm function was analyzed for motility and viability, acrosome integrity, mitochondrial function and phosphatidylserine translocation by flow cytometry before and after cryopreservation. A total of 10 bitches were intravaginal inseminated (IVAI; LN2 frozen-thawed semen = 5 and -80°C-UF frozen-thawed semen = 5). Pregnancy status was confirmed 30 d after IVAI by transabdominal ultrasonography and live born puppies at term were recorded. Sperm function parameters were affected for both freezing protocols. Differences (P < 0.05) were found between freezing and storage methods in most of the parameters of sperm function analyzed, except in the phosphatidylserine translocation. The percentages of pregnancies were not different between the two freezing and storage protocols used. Semen freezing and storage using -80 °C UF is an effective technique for long-term preservation of canine spermatozoa.

Trehalose sustains a higher post-thaw sperm motility than sucrose in vitrified human sperm.

One of the cryopreservation methods that best preserves sperm function is vitrification. However, comparative studies have not been performed to evaluate the effect of nonpermeable cryoprotectors on sperm function for prolonged periods of time post-devitrification. These times are necessary, especially in in vitro fertilisation and intrauterine insemination, for gamete interaction and then fertilisation to occur, while maintaining motility to arrive at the fertilisation site. In this study, sucrose (.25 m) and trehalose (.1 and .05 m) were compared in essential parameters like motility and plasma membrane integrity for 12 hr. Post-devitrification sperm motility using .1 m trehalose was 68.9%, higher than that obtained with .05 m trehalose (59.9%, p < .0081) and .25 m sucrose (57.9%, p < .0002). Similar results were obtained at 6 and 12 hr with .1 m trehalose (58.0% and 42.3% respectively) compared to .05 m trehalose (p < .0184 and p < .033) and .25 m sucrose (p < .0001 and p < .0012).There was no difference between .25 m sucrose and .05 m trehalose. Membrane integrity was best preserved at time 0 by .1 m trehalose (p < .05), but there was no significance at 6 and 12 hr compared to sucrose. Our results suggest that for assisted reproduction techniques that require motile spermatozoa for a longer period of time, use of .1 m trehalose is recommended in the sperm vitrification technique.

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.

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.

Effect of short-term exposure of cumulus-oocyte complex to 3-morpholinosydnonimine on in vitro embryo development and gene expression in cattle.

Short-term exposure of gametes to different types of stress might induce stress tolerance in mammalian embryos. The aim of this study was to evaluate the effect of short-term exposure of bovine mature cumulus-oocyte complex (COC) to 3-morpholinosydnonimine (SIN-1) on subsequent in vitro embryo development, embryo quality and relative gene expression. Matured COCs were incubated with SIN-1 (0, 0.1, 1, 10 and 100 µM SIN-1) for 1 hr before in vitro fertilization and zygotes were cultured until Day 7. The cleavage rate at 72 hr did not show any differences among groups. However, the blastocyst rate on Day 7 decreased with all treatments evaluated, with the embryos generated with 10 µM SIN-1 showing the lowest embryo production rate. Embryo quality analysis did not show any differences in total cell number (TCN) or inner cell mass (ICM) among groups. Relative gene expression analysis showed a downregulation of eNOS expression and an upregulation of nNOS expression in all treatments evaluated compared to the control group. Also, a downregulation was observed in some treatments: SOD2 at 0.1 µM; SOD1 at 0.1 and 100 µM; PRDX5 at 0.1, 10 and 100 µM; and NANOG at 10 and 100 µM; and an upregulation of CDX2 expression was observed at 100 µM. The other genes (OCT4, HIF1A, HSPA1A, BCL2A and iNOS) did not show any differences in the relative gene expression. These results suggest that the short-term exposure of mature bovine COCs to SIN-1 does not induce stress tolerance and has no beneficial effect on bovine in vitro embryo production.

Cryopreservation of Atlantic salmon Salmo salar sperm: effects on sperm physiology.

The objective of this study was to determine the effect of freezing on the function in Atlantic salmon Salmo salar spermatozoa. The semen was frozen in Cortland's medium + 1.3M dimethyl sulphoxide + 0.3M glucose + 2% bovine serum albumin (final concentration) in a ratio of 1:3 (semen:cryoprotectant) as the treatment (T) and fresh semen as the control (F). Straws of 0·5 ml of sperm suspension were frozen in 4 cm of N2 L. They were thawed in a thermoregulated bath (40° C). After thawing, the percentage of spermatozoa with fragmented DNA [transferase dUTP (deoxyuridine triphosphate) nick-end labelling (TUNEL)], plasma membrane integrity (SYBR-14/PI) and mitochondrial membrane potential (ΔΨMMit, JC-1) were evaluated by flow cytometry and motility was evaluated by optical microscope under stroboscopic light. The fertilization rates of the control and treatment semen were tested at a sperm density of 1·5 × 10(7) spermatozoa oocyte(-1) , by observation of the first cleavages after 16 h incubation at 10° C. In the cryopreserved semen (T), the mean ± s.d. DNA fragmentation was 4·8 ± 2·5%; plasma membrane integrity 75·2 ± 6·3%; mitochondrial membrane potential 51·7 ± 3·6%; motility 58·5 ± 5·3%; curved line velocity (VCL ) 61·2 ± 17·4 µm s(-1) ; average-path velocity (VAP ) 50·1 ± 17·3 µm s(-1) ; straight-line velocity (VSL ) 59·1 ± 18·4 µm s(-1) ; fertilization rate 81·6 ± 1·9%. There were significant differences in the plasma membrane integrity, mitochondrial membrane potential, motility, fertilization rate, VCL , VAP and VSL compared with the controls (P < 0·05). Also the mitochondrial membrane potential correlated with motility, fertilization rate, VCL and VSL (r = 0·75; r = 0·59; r = 0·77 and r = 0·79, respectively; P < 0·05); and the fertilization rate correlated with VCL and VSL (r = 0·59 and r = 0·55, respectively).

Viability and DNA fragmentation of rainbow trout embryos (Oncorhynchus mykiss) obtained from eggs stored at 4 °C.

In vitro storage of salmonid eggs leads to aging of the cells causing a decline in quality and reducing their capacity to develop and produce embryos. The quality of salmonid embryos is assessed by morphologic analyses; however, data on the application of biomarkers to determine the cell viability and DNA integrity of embryos in these species are limited. The aim of this study was to evaluate the effect on embryo development, viability and DNA fragmentation in the embryonic cells of in vitro storage time at 4 °C of rainbow trout (Oncorhynchus mykiss) eggs. The embryos were obtained by IVF from eggs stored for 0 (control), 48, and 96 hours at 4 °C. At 72 hours after fertilization, dechorionated embryos were examined to determine percentages of developed embryos (embryos with normal cell division morphology), viability (LIVE/DEAD sperm viability kit), and DNA integrity (terminal deoxynucleotidyl transferase [TdT] dUTP nick-end labeling assay). The percentage of developing embryos decreased (P < 0.05) with storage time of the eggs (95.10 ± 2.55; 88.14 ± 4.50; 79.99 ± 6.60 for 0, 48, and 96 hours, respectively). Similarly, cell viability decreased (P < 0.05; 96.07 ± 7.15; 80.42 ± 8.55; 77.47 ± 7.88 for 0, 48, and 96 hours, respectively), and an increase (P < 0.05) in DNA fragmentation in the embryos was observed at 96-hour storage. A positive correlation was found between cell DNA fragmentation and storage time (r = 0.8173; P < 0.0001). The results revealed that terminal deoxynucleotidyl transferase [TdT] dUTP nick-end labeling assay technique is reliable mean to assess the state of the DNA in salmonid embryos and that in vitro eggs storage for 96h reduces embryo development and cell DNA integrity. DNA integrity evaluation constitutes a biomarker of the quality of the ova and resulting embryos so as to predict their capacity to produce good-quality embryos in salmonids, particularly under culture conditions.

High temperature is essential for preserved human sperm function during the devitrification process.

Sperm vitrification is a cryopreservation method based on high-speed freezing by direct exposure of cells in liquid nitrogen (N2L), thereby avoiding the traditional cooling curves of freezing. The objective of this work was to determine the optimal warming temperature for vitrified human spermatozoa in order to maintain their fertilisation potential. Spermatozoa were cryopreserved by direct plunging into N2L and warmed at different temperatures for 5 and 10 s at 38, 40 and 42 °C. Sperm motility was evaluated by the CASA system and the sperm membrane function by HOST test. It was detected that progressive motility of sperm warmed at 38, 40 and 42 °C was 26.4 ± 8.4%; 56.6 ± 16.3% and 65.4 ± 15%, respectively, with statistically significant differences between the temperatures of 38 and 40 °C and 38 and 42 °C (P < 0.05). The plasma membrane function evaluated by HOST test was better preserved at 42 °C (76.3 ± 2.0%) compared to 40 °C (43 ± 2%) and 38 °C (65.6 ± 1.5%). The temperature in the thawing process can affect the motility and plasma membrane integrity and function. The warming at 42 °C for thawed vitrified sperm is the optimum temperature to preserve the sperm physiological parameters.

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.

Effect of short-term semen storage in salmon (Oncorhynchus mykiss) on sperm functional parameters evaluated by flow cytometry.

The short-term storage of salmonid semen is a viable method for in vitro fertilisation. Previous studies have found that short-term storage affects sperm motility, compromising quality and fertilising capacity. However, the functional characteristics of the spermatozoa of O. mykiss during storage time and its relation to the spawning period are little known. This study was designed to evaluate the effects of in vitro short-term storage on sperm functional parameters in O. mykiss, determined by flow cytometry. Semen samples of the first spawning - undiluted (SSD) and diluted (SD) (Storfish(®) 1 : 2v/v; IMV AI solutions, France) - were stored at 4 °C for 14 days. Motility, viability (PMI: plasma membrane integrity) and mitochondrial membrane potential (ΔΨM) were assessed. On the fifth day of storage, spermatozoa showed a motility >70% (SSD: 78.3% versus SD 85.0%), PMI (81.5% SSD/87.2% SD) and ΔΨM (72.5% SSD/SD 80.0%) (P < 0.05). However, a significant decline in the percentage of all functional parameters (P < 0.05) was observed after 5 days of storage for all samples of both undiluted (SSD) and diluted semen. In conclusion, the results here provide new data on O. mykiss sperm quality with respect to in vitro short-term storage evaluated by flow cytometry.

Protective effect of butylated hydroxytoluene on sperm function in human spermatozoa cryopreserved by vitrification technique.

Butylhydroxytoluene (BHT), a synthetic analogue of vitamin E, shows antioxidant and antiviral properties and has been successfully used for mammalian sperm cryopreservation. In this study, BHT was included in a vitrification solution to determine its cryoprotective effect on human spermatozoa. Spermatozoa were selected by swim-up and vitrified in close sealed straw using either a combination of human tubal fluid (HTF), sucrose and BHT 1 mm (VMBHT), or only HTF and sucrose (VM). The optimal concentration of BHT was determined by the observation of preserved progressive sperm motility (PSM) after warming and detection of plasma membrane (PMI), membrane mitochondrial potential (ΔΨm) and DNA integrity. The presence of reactive oxygen species (ROS) was also detected. The PSM was significantly higher in the VMBHT group (80.86 ± 5.41%) compared with the VM group (68.9 ± 3.67%) (P < 0.05). Butylhydroxytoluene significantly preserved DNA integrity (4.0 ± 0.1% versus 6.1 ± 1.6%; P < 0.05) and reduced ROS production (5.5 ± 2.2 versus 8.6 ± 1.8%; P < 0.05). Plasma membrane and ΔΨm showed no statistical differences. One millimolar BHT effectively maintained cell function and due to its antioxidant and antiviral properties could be used in semen cryopreservation of patients with viral infections transmitted by seminal plasma.

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.

Effect of seminal plasma on Atlantic salmon (Salmo salar) sperm vitrification.

This study was designed to test a vitrification method in Atlantic salmon spermatozoa and determine the capacity of seminal plasma (SP) to protect these cells from cryoinjuries. The vitrification medium consisted of a standard buffer for fish spermatozoa (Cortland medium) + 10% DMSO + 2% BSA + 0.13-M sucrose + SP at concentrations of 30% (G30), 40% (G40), or 50% (G50). Fresh sperm was used as a control. To freeze the samples, 30-µL suspensions of spermatozoa from each group were dropped directly into liquid nitrogen. The resulting spheres were placed in cryotubes for storage in liquid nitrogen. The cryotubes with the vitrified spermatozoa were thawed by placing them in a water bath at 37 °C for 45 seconds. After thawing, the following sperm quality parameters were determined by flow cytometry: DNA fragmentation (terminal deoxynucleotidyl transferase dUTP nick end labeling), plasma membrane integrity (SYBR-14/PI, staining technique), and mitochondrial membrane potential (JC-1 staining). An optical microscope was used to assess subjectively sperm motility, whereas fertility was determined by the presence of neurulation using five replicates per treatment in a sample of 30 eggs. Spermatozoa quality variables were preserved best when the highest concentration of SP (50%) was used (DNA fragmentation, 9.2%; plasma membrane integrity, 98.6%; mitochondrial membrane integrity, 47.2%; motility, 44.1%; and fertility, 46.2%).

Identification of fatty acids in canine seminal plasma.

Seminal plasma contains various biochemical components associated with sperm function. However, there is limited information regarding the fatty acid composition of seminal plasma and their effect on sperm. The aim of this study was to identify the fatty acid content in canine seminal plasma using gas chromatography. Twelve ejaculates were studied, the seminal plasma was obtained by centrifugation and then the lipids were extracted, methylated and analysed by chromatography. The total lipids in the seminal plasma were 2.5 ± 0.3%, corresponding to 85% saturated fatty acids (SFA) and 15% unsaturated fatty acids (UFA). The greatest proportions of SFA were palmitic acid (30.4%), stearic acid (23.4%) and myristic acid (5.3%) and of UFA oleic acid (9.0%). Therefore, the protocols and techniques used enabled the identification of 18 different fatty acids in canine seminal plasma, which constitutes a good method to evaluate and quantify the fatty acid profile in this species.