Spermatological characteristics and effects of cryopreservation in Lebranche mullet spermatozoa (Mugil liza Valenciennes, 1836): First report of ultra-rapid freezing.

The present study investigated the spermatological characteristics of raw semen of Lebranche mullet (Mugil liza), namely pH, and sperm density, and motility; and subsequently evaluated the effects of different times of exposure to cryoprotectants, and the application of an ultra-rapid freezing protocol, on sperm motility and plasma membrane integrity. Semen samples were analyzed undiluted (control) and diluted 1:50 v/v in CF-HBSS + 10% Dimethyl sulfoxide + 30% Ethylene glycol + 94.58 gL-1 Trehalose dehydrate (n = 15). Two treatments - diluted semen samples in cryoprotective medium without ultra-rapid freezing (T1), and diluted semen in cryoprotective medium with ultra-rapid freezing (T2) - were evaluated at 0, 2, 4, 6 and 8 min. The frozen samples were thawed at 37ºC for 60 s. The spermatological characteristics recorded for the semen were: pH: 7.57 ± 0.21; sperm density: 30.4 ± 2.9 × 109 sperm mL-1; motility: 82 ± 4.9%. Sperm motility presented differences after 2 min exposure to cryoprotectants (70.0 ± 2.7%) and ultra-rapid freezing (66.5 ± 5.8%) compared to the control group (98.5 ± 1.9% and 98.5 ± 2.1%, respectively; p < 0.05). On the other hand, the plasma membrane integrity of the spermatozoa after 2 min exposure to cryoprotectants (64.0 ± 8.6%) and ultra-rapid freezing (62.5 ± 5.2%) presented no differences compared to the control group (69.5 ± 3.9% and 70.0 ± 3.5%, respectively p > 0.05); however, differences were observed in the parameters evaluated after longer exposure and cryopreservation times. This is the first report evaluating the effects of different times of exposure to cryoprotectants and direct ultra-rapid freezing in liquid nitrogen on Mugil liza sperm. Our results demonstrated the protocol of sperm ultra-freezing is safe within a time´s window of 2 min of exposure to cryoprotectants, after which a toxicity effect on sperm can be observed.

Treatment patterns and intensification within 5 year of follow-up of the first-line anti-TNFα used for the treatment of IBD: Results from the VERNE study.

BACKGROUND: Anti-TNFα represent one of the main treatment approaches for the management of inflammatory bowel diseases (IBD). Therefore,the evaluation of their treatment patterns over time provides valuable insights about the clinical value of therapies and associated costs. AIMS: To assess the treatment patterns with the first anti-TNFα in IBD. METHODS: Retrospective, observational study. RESULTS: 310 IBD patients were analyzed along a 5-year follow-up period. 56.2% of Crohn's disease (CD) patients started with adalimumab (ADA), while 43.8% started with infliximab (IFX). 12.9% of ulcerative colitis (UC) patients initiated with ADA, while 87.1% initiated with IFX. Treatment intensification was required in 28.9% of CD and 37.1% of UC patients. Median time to treatment intensification was shorter in UC than in CD (5.3 vs. 14.3 months; p = 0.028). Treatment discontinuation due to reasons other than remission were observed in 40.7% of CD and 40.5% of UC patients, although, in UC patients there was a trend to lower discontinuation rates with IFX (36.6%) than with ADA (66.7%). Loss of response accounted for approximately one-third of discontinuations, in both CD and UC. CONCLUSIONS: Around one-third of IBD biologic-naive patients treated with an anti-TNFα required treatment intensification (earlier in UC) and around 40% discontinued the anti-TNFα due to inappropriate disease control.

Natural infection with Trypanosoma cruzi in three species of non-human primates in southeastern Mexico: A contribution to reservoir knowledge.

The mechanisms of infection and dispersion of Trypanosoma cruzi among animals, especially in the sylvatic environment, are still not entirely clear, and various aspects of the transmission dynamics of this parasite in the sylvatic environment are still unknown. T. cruzi is a parasite with a great biological and genetic diversity that infects a wide variety of hosts, therefore, transmission cycles of this parasite are complex. The objective of this study was to determine the prevalence of T. cruzi infection and analyze the genetic variability of the discrete typing units (DTUs) of the parasite in three non-human primate species (Alouatta palliata, Alouatta pigra, and Ateles geoffroyi) in southeastern Mexico. A total of one hundred sixty-four serum samples (42 samples of A. pigra, 41 samples of A. palliata (free-ranging) and 81 samples of A. geoffroyi (hosted in care centers)) were analyzed for the detection of anti-T. cruzi antibodies by ELISA assays. The seroprevalence of infection was 23.39% in A. palliata, 21.40% in A. pigra and 16.27% in A. geoffroyi. Additionally, presence of parasite DNA was assessed by PCR, and the identification of DTUs was performed by real-time PCR coupled to High Resolution Melting (qPCR-HRM). Different DTUs (TcI, TcII, TcIII, TcV and TcVI) were found in the analyzed monkeys. In addition, infection of monkeys was not associated with age or gender, but it was associated with the species. This study reveals the risk of infection in the study area and that the different DTUs of the parasite can coexist in the same habitat, indicating that T. cruzi transmission in the study area is very complex and involves many ecological factors. However, there is a need for long-term studies of host-parasite interactions to provide a solid understanding of the ecology of these species and to understand the dispersion strategies of T. cruzi.

Molecular detection of Rickettsia species in ticks collected in the Mexico-USA transboundary region.

Zoonotic tick-borne diseases, including those caused by Rickettsia species, continue to have serious consequences for public health worldwide. One such disease that has emerged as a major problem in several countries of the American continent is the Rocky Mountain Spotted Fever (RMSF) caused by the bacterium Rickettsia rickettsii. Several tick species are capable of transmitting R. rickettsia, including Amblyomma cajennense, A. aureolatum, A. imitator, Rhipicephalus sanguineus, Dermacentor andersoni, D. variabilis and possibly A. americanum. Despite previous reports in Mexico linking new outbreaks of RMSF to the presence of these tick species, no robust measures have tackled transmission. In the present study, we amplified R. rickettsii from 109 test DNA samples extracted from ticks collected from several animals and humans of Tamaulipas, Mexico, between November 2015 and December 2017. Our analysis revealed the presence of R. rickettsii in six samples and these findings contribute to a spatial distribution map that is intended to minimize the risk of transmission to humans.

Real-world short-term effectiveness of ustekinumab in 305 patients with Crohn's disease: results from the ENEIDA registry.

BACKGROUND: There are limited data of ustekinumab administered according to the doses recommended in the UNITI studies. AIM: To assess the real-world, short-term effectiveness of ustekinumab in refractory Crohn's disease (CD) METHODS: Multicentre study of CD patients starting ustekinumab after June 2017 at the recommend dose (260, 390 or 520 mg based on weight ~6 mg/kg IV week 0 and 90 mg subcutaneously week 8). Values for Harvey-Bradshaw Index (HBI), C-reactive protein (CRP) and faecal calprotectin (FC) were recorded at baseline and at weeks 8 and 14. Demographic and clinical data, previous treatments, AEs and hospitalisations were documented. Possible predictors of clinical remission were examined. RESULTS: Three hundred and five patients were analysed (≥2 previous anti-TNFα therapies 64% and vedolizumab 29%). At baseline, 217 (72%) had an HBI >4 points. Of these, 101 (47%) and 126 (58%) achieved clinical remission at weeks 8 and 14, respectively. FC levels returned to normal (<250 µg/g) in 46% and 54% of the patients at weeks 8 and 14 respectively. CRP returned to normal (<3 mg/L) in the 35% and 41% of the patients at week 8 and 14 respectively. AEs were recorded in 38, and 40 patients were hospitalised. Intolerance to the most recent anti-TNF agent and fewer previous anti-TNF agents were associated with clinical remission at week 14. Endoscopic severity was associated with poor response. CONCLUSION: This is the first study to show the real-world effectiveness and safety of ustekinumab administered according to the recommended induction regimen in a cohort of highly refractory CD patients.

Prevalence and Factors Associated With Fatigue in Patients With Inflammatory Bowel Disease: A Multicentre Study.

BACKGROUND AND AIMS: The aims of this study were to determine the prevalence of fatigue in patients with inflammatory bowel disease [IBD], to identify the factors associated with fatigue and its severity, to assess the impact of fatigue on quality of life [QoL], and to evaluate the relationship between fatigue and sleep disorders. METHODS: This was a prospective multicentre study conducted at 22 Spanish centres. Consecutive patients followed at IBD Units were included. Fatigue was evaluated with the Fatigue Severity Scale [FSS] and the Fatigue Impact Scale [FIS]. Quality of life and sleep quality were assessed using the IBD Questionnaire-Short Form [IBDQ-9] and the Pittsburgh Sleep Quality Index [PSQI], respectively. RESULTS: A total of 544 consecutive adult IBD patients were included [50% women, mean age 44 years, 61% Crohn's disease]. The prevalence of fatigue was 41% (95% confidence interval [CI] = 37-45%). The variables associated with an increased risk of fatigue were: anxiety [OR = 2.5, 95% CI = 1.6-3.7], depression [OR = 2.4, 95% CI = 1.4-3.8], presence of extraintestinal manifestations [EIMs] [OR = 1.7, 95% CI = 1.1-2.6], and treatment with systemic steroids [OR = 2.8, 95% CI = 1.4-5.7]. The presence of EIMs [regression coefficient, RC = 8.2, 95% CI = 2.3-14.2], anxiety [RC = 25.8, 95% CI = 20.0-31.5], depression [RC = 30.6, 95% CI = 24.3-37.0], and sleep disturbances [RC = 15.0, 95% CI = 9.3-20.8] were associated with severity of fatigue. Patients with fatigue had a significantly decreased IBDQ-9 score [p < 0.001]. CONCLUSIONS: The prevalence of fatigue in IBD patients is remarkably high and has a negative impact on QoL. Therapy with systemic steroids is associated with an increased risk of fatigue. The severity of fatigue is associated with anxiety, depression, sleep disorders, and the presence of EIMs. Fatigue was not associated with anaemia, disease activity or anti-TNF therapy.

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.

Post-operative morbidity and mortality of a cohort of steroid refractory acute severe ulcerative colitis: Nationwide multicenter study of the GETECCU ENEIDA Registry.

BACKGROUND: Despite the increased use of rescue medical therapies for steroid refractory acute severe ulcerative colitis, mortality related to this entity still remains high. We aimed to assess the mortality and morbidity related to colectomy and their predictive factors in steroid refractory acute severe ulcerative colitis, and to evaluate the changes in mortality rates, complications, indications of colectomy, and the use of rescue therapy over time. METHODS: We performed a multicenter observational study of patients with steroid refractory acute severe ulcerative colitis requiring colectomy, admitted to 23 Spanish hospitals included in the ENEIDA registry (GETECCU) from 1989 to 2014. Independent predictive factors of mortality were assessed by binary logistic regression analysis. Mortality along the study was calculated using the age-standardized rate. RESULTS: During the study period, 429 patients underwent colectomy, presenting an overall mortality rate of 6.3% (range, 0-30%). The main causes of death were infections and post-operative complications. Independent predictive factors of mortality were: age ≥50 years (OR 23.34; 95% CI: 6.46-84.311; p < 0.0001), undergoing surgery in a secondary care hospital (OR 3.07; 95% CI: 1.01-9.35; p = 0.047), and in an emergency setting (OR 10.47; 95% CI: 1.26-86.55; p = 0.029). Neither the use of rescue medical treatment nor the type of surgical technique used (laparoscopy vs. open laparotomy) influenced mortality. The proportion of patients undergoing surgery in an emergency setting decreased over time (p < 0.0001), whereas the use of rescue medical therapy prior to colectomy progressively increased (p > 0.001). CONCLUSIONS: The mortality rate related to colectomy in steroid refractory acute severe ulcerative colitis varies greatly among hospitals, reinforcing the need for a continuous audit to achieve quality standards. The increasing use of rescue therapy is not associated with a worse outcome and may contribute to reducing emergency surgical interventions and improve outcomes.

Sperm characteristics of wild and captive lebranche mullet Mugil liza (Valenciennes, 1836), subjected to sperm activation in different pH and salinity conditions.

In this article we describe basic aspects of the sperm biology of lebranche mullet (Mugil liza) in the wild and in captivity, in particular assessing the effects of salinity (0, 10, 20, 30, 35, 40, 50 and 60 g L-1) and pH (6, 7, 8, 9 and 10) on sperm motility. Our results indicate that the highest percentage of motility was recorded with salinity 34.6 g L-1 (95 ±â€¯10%) and the longest motility time was obtained with a salinity of 34.8 g L-1 (189 ±â€¯15 s). Variations in the salinity between 30 and 35 g L-1 did not produce any significant alterations in sperm motility; however salinities of 20 and 50 g L-1 produced a significant loss of sperm motility. The highest percentage of motility was obtained at pH 8.5 (93 ±â€¯12%), and the longest motility period at pH 8.7 (218 ±â€¯13 s), while pH lower than or equal to 7 and equal to 10 both produced a significant loss in sperm motility. A positive correlation was found between pH/salinity and the motility percentage (R2 = 0.94 and R2 = 0.97) and motility time (R2 = 0.86 and R2 = 0.98). In seminal and morphometric parameters, statistically significant differences were observed in semen volume, sperm density, plasma membrane integrity and sperm morphometry between the groups studied, showing that the characteristics of the fish have a direct influence on sperm quality. The information generated in this research will be useful for developing biotechnology tools for the effective management of Mugil liza gametes.

Long-Term Safety of In Utero Exposure to Anti-TNFα Drugs for the Treatment of Inflammatory Bowel Disease: Results from the Multicenter European TEDDY Study.

OBJECTIVES: The long-term safety of exposure to anti-tumor necrosis factor (anti-TNFα) drugs during pregnancy has received little attention. We aimed to compare the relative risk of severe infections in children of mothers with inflammatory bowel disease (IBD) who were exposed to anti-TNFα drugs in utero with that of children who were not exposed to the drugs. METHODS: Retrospective multicenter cohort study. Exposed cohort: children from mothers with IBD receiving anti-TNFα medication (with or without thiopurines) at any time during pregnancy or during the 3 months before conception. Non-exposed cohort: children from mothers with IBD not treated with anti-TNFα agents or thiopurines at any time during pregnancy or the 3 months before conception. The cumulative incidence of severe infections after birth was estimated using Kaplan-Meier curves, which were compared using the log-rank test. Cox-regression analysis was performed to identify potential predictive factors for severe infections in the offspring. RESULTS: The study population comprised 841 children, of whom 388 (46%) had been exposed to anti-TNFα agents. Median follow-up after delivery was 47 months in the exposed group and 68 months in the non-exposed group. Both univariate and multivariate analysis showed the incidence rate of severe infections to be similar in non-exposed and exposed children (1.6% vs. 2.8% per person-year, hazard ratio 1.2 (95% confidence interval 0.8-1.8)). In the multivariate analysis, preterm delivery was the only variable associated with a higher risk of severe infection (2.5% (1.5-4.3)). CONCLUSIONS: In utero exposure to anti-TNFα drugs does not seem to be associated with increased short-term or long-term risk of severe infections in children.

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.

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.

Long-Term Efficacy and Safety of Cyclosporine in a Cohort of Steroid-Refractory Acute Severe Ulcerative Colitis Patients from the ENEIDA Registry (1989-2013): A Nationwide Multicenter Study.

OBJECTIVES: To determine the efficacy and safety of cyclosporine (CyA) in a large national registry-based population of patients with steroid-refractory (SR) acute severe ulcerative colitis (ASUC) and to establish predictors of efficacy and adverse events. METHODS: Multicenter study of SR-ASUC treated with CyA, based on data from the ENEIDA registry. SR-ASUC patients treated with infliximab (IFX) or sequential rescue therapy (CyA-IFX or IFX-CyA) were used as comparators. RESULTS: Of 740 SR-ASUC patients, 377 received CyA, 131 IFX and 63 sequential rescue therapy. The cumulative colectomy rate was higher in the CyA (24.1%) and sequential therapy (32.7%) than in the IFX group (14.5%; P=0.01) at 3 months and 5 years. There were no differences in early and late colectomy between CyA and IFX in patients treated after 2005. 62% of patients receiving CyA remained colectomy-free in the long term (median 71 months). There were no differences in mortality between CyA (2.4%), IFX (1.5%) and sequential therapy (0%; P=0.771). The proportion of patients with serious adverse events (SAEs) was lower in CyA (15.4%) than in IFX treated patients (26.5%) or sequential therapy (33.4%; P<0.001). This difference in favor of CyA was maintained when only patients treated after 2005 were analyzed. CONCLUSIONS: Treatment with CyA showed a lower rate of SAE and a similar efficacy to that of IFX thereby supporting the use of either CyA or IFX in SR-ASUC. In addition, the risk-benefit of sequential CyA-IFX for CyA non-responders is acceptable.

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.

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).

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.

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.

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%).