Influences of a diet supplemented with linseed oil and antioxidants on quality of equine semen after cooling and cryopreservation during winter

Seasonal changes in the reproductive physiology of stallions contribute to a decrease in the quality of frozen-thawed semen during late winter. Changes in the lipid composition of the sperm plasma membrane may contribute to this phenomenon. In the present study, we have, therefore, investigated the effects of adding linseed oil (LO) in combination with antioxidants to the diet of breeding stallions on the motility and membrane integrity of cooled–stored and cryopreserved semen. Starting in November, the diet of LO stallions (n = 6) but not control (C) stallions (n = 5) was supplemented with LO (100 mL once daily) plus an antioxidant (Myostem Protect; Audevard, Clichy, France) for a total of 84 days. Before (November) and at the end of this period (February), ejaculates were processed for cryopreservation (n = 3 ejaculates per stallion) and cooled shipping at 5 °C. Frozen-thawed and cooled–shipped semen was sent to the laboratory for computer-assisted semen analysis of total motility, progressive motility, and velocity parameters (average path velocity [VAP], curved line velocity [VCL], and straight-line velocity [VSL]) and evaluation of membrane integrity. The quality of frozen-thawed semen decreased (P < 0.05) from November (e.g., total motility LO 69 ± 3% and C 67 ± 3%) to February (total motility: LO 55 ± 4% and C 59 ± 3%) independent of treatment (P > 0.05). A decrease in the velocity parameters VAP, VCL, and VSL was more pronounced in LO stallions than in C stallions (e.g., VSL: November LO 67 ± 1 μm/s, C 64 ± 2 μm/s; February LO 59 ± 2 μm/s, C 63 ± 2 μm/s; interaction month by treatment, P < 0.05). In cooled–stored semen, total motility, progressive motility, and membrane integrity were lower in February than in November (P < 0.001 for all parameters). Supplementation of the diet with LO and antioxidants attenuated this decrease (e.g., Day 1 of cooled storage = 24 hours after semen collection: total motility in November LO 88 ± 1% and C 87 ± 3%; in February LO 83 ± 2% and C 73 ± 11%; interaction month by treatment: P < 0.05). Velocity parameters VAP, VCL, and VSL were significantly lower in February than in November (P < 0.001), but this decrease was not affected by treatment. In summary, dietary supplementation of stallions with LO plus antioxidants attenuated a decline in motility and membrane integrity of cooled–stored stallion semen during winter. This may improve the fertility of cooled–shipped semen. In contrast, the treatment did not counteract the decrease in quality of frozen-thawed semen that occurs in late winter.     

Consequences of adding gum Arabic as a cryoprotectant on motility and viability of frozen stallion semen

A trial was conducted to check effect of adding gum Arabic (GA) instead of egg yolk (EY) as a cryoprotectant for stallion sperm. Two experiments were designed; experiment I tested adding 3 levels of nonheated GA (i.e., 3, 6 and 9 g/100 mL diluents) in HF-20 extender. However, in experiment II the same levels were tested except that GA was heated at 80 °C for 60 min. HF-20 containing 10% of EY was used as control. In experiment I, sperm frozen in HF-20 containing nonheated GA exhibited lower percentages of motile sperm, progressively motile sperm and sperm with intact plasma membranes, vitality rate, and acrosome integrity after cooling or after deep freezing. Frozen semen in HF-20 containing 3–6% of preheated GA in experiment II maintained sperm motility at 46–50% and elevated progressive motility at 27%. The semen diluted in preheated GA (6%) and frozen exhibited a fertility rate of 40% (2/5). A similar fertility rate (40%) was found in the control semen (i.e. 10%) compared to those that were inseminated with frozen semen in preheated 3% GA (20%, 1/5). These results suggest that preheated GA could be used as an alternative cryoprotectant for cryopreserving stallion sperm.     

Quality of chilled and cold-stored (5 °C) canine spermatozoa submitted to different rapid cooling rates

The aim of this study was to evaluate the sperm quality in chilled canine semen using di?erent cooling rates from room temperature (23 °C) to 5 °C and subsequently cold-stored at 5 °C for up to 96 hours. In experiment 1, semen samples from five dogs were pooled, diluted in Tris-fructose-citrate extender with 20% egg yolk and split into four aliquots that were chilled to 5 °C using di?erent cooling rates of 2.25, 0.9, 0.45, and 0.2 (control) °C/min. In experiment 2, semen from five dogs was processed individually as described above and split into two aliquots that were chilled to 5 °C using rates of either 2.25 °C/min or 0.2 °C/min. In both experiments, the sperm quality (i.e., sperm motility and viability) was evaluated before cooling and after 0, 24, 48, 72, and 96 hours of storage at 5 °C. The total motility, progressive motility, and quality of movement parameters were assessed using computer-assisted analysis system, and the percentage of viable spermatozoa was determined using ?ow cytometry (H-42/PI//FITC-PNA). The cooling rate did not in?uence the sperm quality parameters at any of the evaluation times. All evaluated males showed the same response to chilling semen at a rapid cooling rate. Storage time negatively in?uenced (P < 0.05) sperm motility, regardless of the cooling rate used. In conclusion, canine sperm could be chilled and stored for 96 hours at 5 °C in a Tris-fructose extender with 20% egg yolk using rapid cooling rates, with values for sperm quality similar to those from a conventional protocol.     

Sperm quality of Colossoma macropomum after room‐temperature and cold storage

The objective of this study was to evaluate the effects of cold and room‐temperature storage on the quality of Colossoma macropomum sperm. The experiment was carried out in December (end of Spring), in Nova Mutum‐MT, Brazil, involving nine C. macropomum males (4 years old; 6.4 ± 1.5 kg average weight). The fish were selected and transferred to masonry tanks (4 m³) in a laboratory (water renewal rate: 10 L/s; average water temperature: 28°C). Subsequently, reproduction was induced using 2.5 mg of crude carp pituitary extract/kg and the semen was harvested 240 degree hours after hormonal induction. The following sperm characteristics were analyzed every 5 hr using Image J/casa software: total motility (MOT), curvilinear velocity (VCL), average path velocity (VAP), straight‐line velocity (VSL), straightness of sperm path (STR), wobble (WOB), progressive motility (PROG), beat cross frequency (BCF) and total number of spermatozoa (NSPZ). A fresh sample of semen from each animal was kept at room temperature (25.3 ± 1.2°C). For analysis of cooled semen, syringes were kept in cooling boxes at an average temperature of 16.9 ± 2.1°C. The reduction (p < 0.05) of MOT in semen kept at room temperature occurred at 10 hr (13.95%); in cooled semen, however, MOT declined at 15 hr (76.87%). At 15 hr, there was practically no MOT in the semen kept at room temperature (0.20%), whereas in the cooled semen this situation was observed only at 35 hr (2.91%) The MOT of cooled sperm was higher (p < 0.05) at all times (except zero time), compared with the semen maintained at room temperature. At 15 hr, the cooled spermatozoa showed higher (p < 0.05) VCL (142.18 μm/s) and BCF (29.72 Hz) than those maintained at room temperature (VCL: 51.18 μm/s; BCF: 19.57 Hz). After 15 hr, only the cooled sperm showed quality. In conclusion, semen cooling allows for extending the viability of C. macropomum spermatozoa from 5 to 10 hr without compromising their quality in most characteristics. At 15 and 25 hr of cooling, sperm viability is still observed, though with decreased quality.     

Pellet cryopreservation for chicken semen: Effects of sperm working concentration,cryoprotectant concentration,and equilibration time during in vitro processing

The aim of the study was to standardize the pellet cryopreservation procedure for chicken semen. Mericanel della Brianza male chicken breeders (Italian breed) were used. Pooled semen samples were processed according to the following conditions: (1) dilution in prefreezing extender to 1 versus 1.5 bill cells/mL sperm working concentration (SWC); (2) 6% versus 9% dimethyl acetamide (DMA) concentration (DMAco); (3) 1 versus 30 minutes DMA equilibration (DMAeq) at 4 °C. Sperm viability and motility were assessed in semen (four replicates/treatment) soon after collection (time 0), after DMAeq (time D), and after freezing/thawing (time FT). The recovery rates (%) of viable and motile sperm after freezing/thawing were also calculated. The low SWC (1 bill/mL) and the low DMAco (6%) indicated a positive significant effect on the proportion of motile sperm (1 bill/mL = 53% vs. 1.5 bill/mL = 48%; 6% DMA = 55% vs. 9% DMA = 47%). Very short DMAeq (1 minute) did not significantly change sperm viability during processing (from time 0 to time D) before freezing whatever the DMAco, and, in contrast, the longer DMAeq showed a significant negative effect on sperm viability. The highest proportion of motile sperm was recorded in semen samples diluted to 1 bill/mL and added with 6% DMA; in this condition, DMAeq had no effect (57% 1 minute and 61% 30 minutes). Increasing SWC to 1.5 bill/mL and adding again 6% DMA, a significant effect of DMAeq was observed, and the higher proportion of motile sperm (58% vs. 43%) was recorded after 1 minute DMAeq. A general decrease in sperm motility was shown in semen samples with 9% DMA (47% vs. 55%), and different conditions in SWC and DMAeq were not effective in the prevention of such decrease.     

Effects of dilution and centrifugation on the survival of spermatozoa and the structure of motile sperm cell subpopulations in refrigerated Catalonian donkey semen

The aim of this work was to study the effects of dilution and centrifugation (i.e., two methods of reducing the influence of the seminal plasma) on the survival of spermatozoa and the structure of motile sperm cell subpopulations in refrigerated Catalonian donkey (Equus asinus) semen. Fifty ejaculates from nine Catalonian jackasses were collected. Gel-free semen was diluted 1:1, 1:5 or 1:10 with Kenney extender. Another sample of semen was diluted 1:5, centrifuged, and then resuspended with Kenney extender until a final dilution of 25 × 10⁶ sperm/ml was achieved (C). After 24 h, 48 h or 72 h of refrigerated storage at 5 °C, aliquots of these semen samples were incubated at 37 °C for 5 min. The percentage of viable sperm was determined by staining with eosin-nigrosin. The motility characteristics of the spermatozoa were examined using the CASA system (Microptic, Barcelona, Spain). At 24 h, more surviving spermatozoa were seen in the more diluted and in the centrifuged semen samples (1:1 48.71%; 1:5 56.58%, 1:10 62.65%; C 72.40%). These differences were maintained at 48 h (1:1 34.31%, 1:5 40.56%, 1:10 48.52%, C 66.30%). After 72 h, only the C samples showed a survival rate of above 25%. The four known donkey motile sperm subpopulations were maintained by refrigeration. However, the percentage of motile sperms in each subpopulation changed with dilution. Only the centrifuged samples, and only at 24 h, showed exactly the same motile sperm subpopulation proportions as recorded for fresh sperm. However, the 1:10 dilutions at 24 and 48 h, and the centrifuged semen at 48 h, showed few variations compared to fresh sperm. These results show that the elimination of seminal plasma increases the survival of spermatozoa and the maintenance of motility patterns.The initial sperm concentration had a significant (P < 0.05) influence on centrifugation efficacy, but did not influence the number of spermatozoa damaged by centrifugation. In contrast, the percentage of live spermatozoa in the fresh semen significantly influenced the number of spermatozoa damaged by centrifugation, but not centrifugation efficacy.     

Effect of Uremia on Semen Quality and Reproductive Function in Humans

In this study, we sought to evaluate the effect of uremia on semen quality and reproductive function in humans. For this purpose, 53 end-stage uremic patients were randomly selected. The semen samples were produced by masturbation. Fertility index (FI) was calculated according to the following formula: sperm density (×10⁶/ml) × sperm motility (%) × normal sperm morphology rate (% per 10,000). The semen samples of uremic patients were compared with those of fertile and infertile males. The results show that three patients failed to produce semen. There were no sperm found in four semen samples. The sperm motility, survival rate, sperm density, and normal sperm morphology rate of the remaining 46 patients were found to be significantly lower than those of controls. The uremic patients had the FI of 0.68(2.08) which was obviously lower than that of fertile 7.7(13.51) and infertile 4.13(5.77) males. It was, therefore, concluded that uremia caused a significant decline in sperm quality and reproductive function which resulted in consequential infertility in humans.     

Effects of mint,thyme, and curcumin extract nanoformulations on the sperm quality,apoptosis, chromatin decondensation,enzyme activity,and oxidative status of cryopreserved goat semen

Goat semen cryopreservation is a challenging process as it results in reduced motility, vitality, and fertility of spermatozoa after freezing. In this study, we evaluated the effects of different herbal extract nanoformulations (NFs) [mint (MENFs), thyme (TENFs), and curcumin (CENFs)], supplemented at either 50 or 100 μg into Tris-extender on the cryopreserved goat semen quality. The hydrothermal squeezing method was used for the preparation of the NFs extracts. The morphological evaluation of the NFs extracts was conducted by transmission electron microscopy. All NFs supplements improved (p < 0.05) the progressive motility, vitality, and plasma membrane integrity of sperm compared with the control extender after equilibration (5 °C for 2 h) and thawing (37 °C for 30 s), but had no effect on sperm abnormality and acrosome integrity. All NFs supplements decreased (p < 0.05) the apoptosis, malondialdehyde level, and chromatin decondensation of sperm cells, while increased (p < 0.05) the total antioxidant capacity and catalase activity in the frozen/thawed extender. Particularly, CENFs at a level of 100 μg showed improvement of sperm parameters and antioxidant status during cryopreservation of goat semen more than TENFs and MENFs. The CENFs improved the quality of goat spermatozoa in post-thawed semen in terms of preventing cryodamage and promoting the cryotolerance of spermatozoa when compared with TENFs and MENFs. Therefore, supplementation of Tris-extender with CENFs could enhance goat semen processing during cryopreservation.     

Can amides be alternative cryoprotectors for the preservation of feline semen?

Sperm cryopreservation is a tool for the conservation of the genetic material of animals of genetic importance or for species preservation. In the case of domestic cats, this can be used to generate information about seminal harvest, evaluation and preservation, which is especially important due to its applicability to wild felids. This study evaluated seminal samples harvested by urethral catheterisation from 13 adult domestic cats. Samples were cryopreserved with experimental groups of extenders were defined by the penetrating cryoprotectant: 6% glycerol (GLY6%), 3% dimethylacetamide (DMA3%) and 3% dimethylformamide (DMF3%). The samples were thawed and evaluated by conventional microscopy and by computer-assisted sperm analysis (CASA). The structural and functional membrane integrity was assessed by supravital tests (EOS), hypoosmotic swelling tests (HOST) and flow cytometry (FC). There was a correlation (P < 0.05) between total motility and EOS (r = 0.54), HOST and FC (r = −0.62) and total motility and flow cytometry (r = 0.63), indicating that these are complementary parameters that increase the accuracy of the feline sperm quality evaluation post-thaw. The results regarding the structural and functional integrity of the sperm plasma membrane did not differ (P > 0.05) among groups. However, the DMA3% group had a lower (P < 0.05) percentage of morphological changes in the sperm tail compared to samples cryopreserved with GLY6% and DMF3%. Additionally, DMA3% provided lower values of immobile sperm post-thaw when compared to DMF3%. DMA is an interesting alternative to GLY and superior to DMF for the cryopreservation of feline semen at the studied concentrations.     

Pregnancy rates after artificial insemination with cooled stallion spermatozoa either with or without Single Layer Centrifugation

A successful outcome after artificial insemination with cooled semen is dependent on many factors, the sperm quality of the ejaculate being one. Previous studies have shown that spermatozoa with good motility, normal morphology, and good chromatin integrity can be selected by means of colloid centrifugation, particularly single layer centrifugation (SLC) using species-specific colloids. The purpose of the present study was to conduct an insemination trial with spermatozoa from “normal” ejaculates, i.e., from stallions with no known fertility problem, to determine whether the improvements in sperm quality seen in SLC-selected sperm samples compared with uncentrifuged controls in laboratory tests are reflected in an increased pregnancy rate after artificial insemination. In a multicentre study, SLC-selected sperm samples and uncentrifuged controls from eight stallions were inseminated into approximately 10 mares per treatment per stallion. Ultrasound examination was carried out approximately 16 days after insemination to detect an embryonic vesicle. The pregnancy rates per cycle were 45% for controls and 69% for SLC-selected sperm samples, which is statistically significant (P < 0.0018). Thus, the improvement in sperm quality reported previously for SLC-selected sperm samples is associated with an increase in pregnancy rate, even for ejaculates from stallions with no known fertility problem.     

Dietary omega-3 fatty acids from linseed oil improve quality of post-thaw but not fresh sperm in Holstein bulls

Docosahexaenoic acid (DHA), a member of the n-3 fatty acid family present in fish oil, has several positive effects on bovine sperm, including membrane integrity, motility and viability, as well as cold sensitivity. Our objective was to investigate effects of varying amounts of omega-3 fatty acids from linseed oil, administered orally, on quality of fresh and frozen-thawed bull sperm. Twenty fertile Holstein bulls (874 ± 45.38 kg) were randomly and equally assigned to four groups and received encapsulated (rumen-protected) fats for 12 weeks, as follows: group P, 300 g palm oil; group Pl, 200 g palm oil + 100 g linseed oil; group pL, 100 g palm oil + 200 g linseed oil; and group L, 300 g linseed oil. Sperm quality of fresh and frozen-thawed semen was evaluated by routine assays including sperm motion characteristics (CASA), membrane integrity (eosin-nigrosin), membrane activity (hypo-osmotic swelling test; HOST) and malondialdehyde (MDA) content. There were no significant differences among groups in semen volume, sperm concentration or sperm quality parameters in fresh semen. However, after freezing-thawing, total and progressive motility in group P (59.61 ± 1.95 and 40.19 ± 2.48%, respectively; LSM ± SEM) were lower (P < 0.05) than in groups Pl (66.06 ± 1.95 and 47.53 ± 2.48%), pL (65.67 ± 1.95 and 47.48 ± 2.48%) and L (65.36 ± 1.95 and 47.62 ± 2.48)%, with no significant differences among the latter three groups. Furthermore, membrane integrity (eosin-nigrosin) and activity (HOST) were lower (P < 0.05) in group P (55.79 ± 2.15 and 42.19 ± 2.17%) compared to groups Pl (62.73 ± 2.15 and 48.93 ± 2.17%), pL (64.06 ± 2.15 and 50.01 ± 2.17%) and L (64.47 ± 2.15 and 49.68 ± 2.17%), with no significant differences among the latter three. Furthermore, there were more (P < 0.05) morphologically abnormal sperm in group P (25.99 ± 1.62%) than in groups Pl, PL and L (21.55 ± 1.62, 21.69 ± 1.62 and 20.90 ± 1.62%). In conclusion, feeding Holstein bulls 100–300 g linseed oil daily improved sperm cryotolerance.     

Morphological and morphometric evaluation of silver barb,Barbodes gonionotus (Bleeker, 1849) sperm supplemented with antibiotics

Aim of this study was to evaluate sperm morphology of silver barb, Barbodes gonionotus, sperm and describe the effect of antibiotics on morphological characteristics of the sperm using an ASMA plug‐in. The experiment was done at the room temperature (25°C) and divided into four treatments in three replicates: (i) freshly collected semen, (ii) extended semen (control), (iii) extended semen supplemented with 0.5% penicillin‐streptomycin (PS), and (iv) extended semen supplemented with 0.5% penicillin‐gentamicin (PG). Silver barb sperm comprised three main compartments: a circular head with no acrosome, a midpiece, and a single flagellum. Addition of 0.5% PS had no detrimental effects on sperm morphometry, except flagellum width. Administration of 0.5% PG affected sperm morphology in two distinct ways: (i) intact sperm (76.92 ± 5.84% of total sperm) except for flagellum width, and (ii) severe morphological damage (23.08 ± 2.67%).     

Fertility and uterine hemodynamic in cows after artificial insemination with semen assessed by fluorescent probes

Fluorescent probes (propidium iodide, Hoechst 33342, fluorescein isothiocyanate–conjugated Pisum sativum agglutinin, and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide) were used to simultaneously evaluate the integrity of plasma and acrosomal membranes as well as mitochondrial membrane function in cryopreserved bovine semen and to verify its influence on fertility and postinsemination uterine vascularization. One hundred eighty-two Nellore cows were distributed for artificial insemination (AI) using semen batches separated according to the cell percentage presenting intact plasma membrane, intact acrosome, and high mitochondrial function (IPIAH): group G (44.5% IPIAH, n = 68), group M (23.0% IPIAH, n = 56), and group R (8.5% IPIAH, n = 58). The uterine hemodynamic was evaluated by Doppler sonogram in three periods: 30 hours before AI, 4 and 24 hours after AI were considered the resistance index and the uterine vascularization score. The pregnancy rate of group G (64.7%) was greater (P > 0.05) compared with group R (36.2%), but both did not differ from group M (50.0%). There was no effect (P > 0.05) of semen quality on uterine vascularization. Greater vascularization was noticed 4 hours after AI than 30 hours before and 24 hours after AI. Semen evaluation using fluorescent probes contributes to predicting fertilizing potential of semen. The use of semen with less percentage of IPIAH sperm does not alter uterine hemodynamic in cows.     

Changes in the nutritional quality of decaying leaf litter in a stream based on fatty acid content

We examined the nutritional quality of decaying leaf litter in a third-order forested stream, using measurements of fatty acid (FA) composition over time. We measured changes in concentrations of total, polyunsaturated, microalgal, and microbial marker FAs in mixed-species leaf packs in spring and autumn and effects of including/excluding macroinvertebrates. Initial concentrations of total FAs in litter were significantly less in spring (5.2 mg/g) than in autumn (6.9 mg/g; F = 6.3; P = 0.03), but total FA concentrations in litter placed in the stream declined significantly over 120 days in both spring (62%; F = 10.9; P < 0.001) and autumn (56%; F = 19.4; P = 0.0001). Quantities of most FAs declined at a greater rate than that of bulk leaf matter. The presence or absence of macroinvertebrates (5 mm vs. 250 μm mesh) had no effect on FA concentration or composition of decomposing litter. Omega-3 polyunsaturated FAs were either nearly absent (20:5ω3) or depleted preferentially over other FAs (18:3ω3). During decomposition the polyunsaturated FA linoleic acid (18:2ω6, common in fungi), declined in concentration more rapidly than other FAs in the spring, but in autumn declined at slower rates, perhaps suggesting greater fungal activity in autumn. Quantities of bacterial (e.g., 16:1ω7) and fungal (e.g., 18:1ω9) FA markers increased over time in autumn (and 16:1ω7 also in spring). Our data provide no evidence for increasing nutritional FA quality of litter during decay and microbial colonization, based on total and polyunsaturated FAs, despite measured increases in bacterial and fungal FA over time. Routine measurements of FA composition of litter could provide insights into the nutrition of allochthonous matter and the importance of fungi and bacteria during decomposition.     

Pre-incubation prior to semen processing and the subsequent effect on the quality of fresh-cooled and cryopreserved ram semen

For artificial insemination (AI) in the pig, semen is routinely maintained at room temperature for 2–4 h prior to extending—to reduce the cooling damage to sperm during cryopreservation. In the sheep industry, however, semen is diluted and cooled immediately after collection. This trial evaluated the effect of a 4 h pre-incubation period for semen at room temperature on the subsequent quality parameters of ram sperm prepared for AI. Immediately following collection, ram semen was divided in 2 aliquots—one was left undiluted for 4 h at room temperature (20 °C; pre-incubation) and the other (control) was diluted with an egg-yolk-based extender and either cooled to 5 °C (n = 8 different ejaculates) for short-term fresh conservation or cryopreserved (n = 6 different ejaculates). After 4 h at room temperature, the pre-incubated semen was then diluted and either cooled to 5 °C or cryopreserved, as was the control. Sperm motility, viability and chlortetracycline (CTC) pattern distribution of the pre-incubated semen were compared to the control. For fresh semen conserved at 5 °C, total sperm motility and the proportion of CTC pattern F sperm (referring to non-capacitated, non-acrosome reacted cells) were reduced by the 4 h incubation at room temperature, compared to the control. The effect of pre-incubation at room temperature was more evident in the cryopreserved semen in terms of total and progressive sperm motility, with the viability being reduced following pre-incubation. For the cryopreserved semen, the percentage of CTC pattern F sperm declined, while the pattern of AR sperm (referring to acrosome-reacted cells) increased, compared to the controls. In conclusion, pre-incubation of ram semen for 4 h at room temperature prior to preparation for AI is not beneficial to the subsequent functionality of the sperm. Furthermore, this pre-incubation period is more harmful to frozen-thawed than to fresh-cooled sperm.     

Effects of age and environmental factors on semen production and semen quality of Austrian Simmental bulls

More than 90% of the breeding stock of Austrian dual purpose Simmental cows is artificially inseminated. Knowledge of factors affecting sperm production and semen quality is of importance with regard to reproductive efficiency and thus genetic improvement as well as for the productivity and profitability of AI centers. Hence, semen data from two Austrian AI centres collected in the years 2000 and 2001 were evaluated. In total, 3625 and 3654 ejaculates from 147 and 127 AI bulls, respectively, were analysed regarding ejaculate volume, sperm concentration, percentage of viable spermatozoa in the ejaculate, total spermatozoa per ejaculate and motility. Effects accounted for were the bull (random), age of bull, collection interval, number of collection on collection day, bull handler, semen collector, temperature on day of semen collection, in the course of epididymal maturation (average temperature of days 1-11 before collection) and during spermatogenesis (average temperature of days 12-65 before collection). Age of bull significantly affected all traits (P<0.01 to P<0.001) except motility score in center 2. Ejaculate volume and total number of spermatozoa increased with age of bull while sperm concentration was lower in higher age classes (center 1). The collection team was also found to significantly influence semen quality traits. With increasing collection interval ejaculate volume and total number of spermatozoa increased significantly (P<0.05 to P<0.001) while collection intervals between 4-9 days and 1-6 days were superior with regard to sperm concentration and percentage of viable spermatozoa, respectively (P<0.10 to P<0.001). First ejaculates were superior with respect to ejaculate volumes, sperm concentrations and total number of spermatozoa per ejaculate (P<0.001). Temperature, either on day of semen collection or during epididymal maturation or spermatogenesis, had important but inconsistent effects on semen production and sperm quality. Overall, however, ambient temperatures in the range of 5-15 degrees C were found to be optimal for semen production.     

Validation of merocyanine 540 staining as a technique for assessing capacitation-related membrane destabilization of fresh dog sperm

The aim of this study was to determine whether flow cytometric evaluation of combined merocyanine 540 and Yo-Pro 1 (M540-YP) staining would identify viable dog sperm that had undergone membrane stabilization known to be associated with capacitation in other species, and whether such destabilization is detected earlier than when using the tyrosine phosphorylation and ethidium homodimer (TP-EH) stain combination with epifluorescence microscopy. Semen from nine dogs was collected and incubated in parallel in bicarbonate-free modified Tyrode's medium (−BIC), medium containing 15 mM bicarbonate (+BIC), dog prostatic fluid, and in PBS. Aliquots for staining were removed at various time points during incubation of up to 6 hours. Staining with M540-YP allowed the classification of dog sperm as viable without destabilized membranes, viable with destabilized membranes, nonviable without destabilized membranes, or nonviable with destabilized membranes. The percentage of viable sperm detected using EH (83.5 ± 1.37%; mean ± SEM) was higher than when using YP (66.7 ± 1.37%: P < 0.05; n = 54 semen samples). On the other hand, M540-YP identified a higher percentage of viable sperm with destabilized membranes than TP-EH (75 ± 1.76% vs. 35 ± 1.70%: P < 0.05; n = 54 semen samples). Staining with M540-YP indicated a rapid increase in the percentage of viable sperm with destabilized membranes, reaching a maximum during the first 30 minutes of incubation in +BIC. For all other treatments (i.e., −BIC, prostatic fluid, and PBS), the peak in the percentage of viable sperm with destabilized membranes was reached as much as 90 to 210 minutes later than incubation in +BIC. The lowest percentage of viable sperm showing signs of capacitation was recorded during incubation in PBS. We conclude that YP identifies sperm committed to cell death earlier than EH, and that the M540-YP stain combination identifies membrane destabilization known to be associated with capacitation in other species earlier than the TP-EH stain combination.     

Factors impacting equine sperm recovery rate and quality following cushioned centrifugation

Two experiments were conducted to investigate modifications in cushioned centrifugation of stallion semen. Specifically, the effects of tube type, centrifugation medium, cushion type, and centrifugation force on post-centrifugation sperm recovery rate and quality were evaluated. In Experiment 1, sperm recovery rate was higher (P<0.05) in conventional plastic conical-bottom tubes (103%) than in newly developed glass nipple-bottom tubes (96%) following cushioned centrifugation; however, several measures of semen quality (i.e., % total motility [MOT], % progressive motility [PMOT], curvilinear velocity, and average-path velocity) yielded higher values following centrifugation in nipple-bottom tubes (P<0.05). Sperm recovery rate following cushioned centrifugation was similar between semen previously diluted in optically clear centrifugation extender (100%) and semen diluted in opaque centrifugation extender (100%); however, MOT and PMOT were higher in semen subjected to cushioned centrifugation in opaque extender (P<0.05). An extender by tube-type interaction was not detected for recovery rate or post-centrifugation semen quality. In Experiment 2, sperm recovery rate following cushioned centrifugation in nipple-bottom tubes was similar when forces of 400xg or 600xg were applied (90 and 90%, respectively; P>0.05), and no resulting differences in semen quality were detected between these treatment groups (P>0.05). The type of iodixanol cushion medium used (i.e., OptiPrep, Eqcellsire Component B, or Cushion Fluid did not impact post-centrifugation semen quality, based on the laboratory values measured (P>0.05). In conclusion, cushioned centrifugation of stallion semen in either conical-bottom or nipple-bottom tubes yielded a high sperm harvest, while maintaining sperm function. An optically opaque extender, commonly used in the equine breeding industry, can be used to achieve this goal.     

Radon and risk of death from cancer and cardiovascular diseases in the German uranium miners cohort study: follow-up 1946–2003

Data from the German uranium miners cohort study were analyzed to investigate the radon-related risk of mortality from cancer and cardiovascular diseases. The Wismut cohort includes 58,987 men who were employed for at least 6 months from 1946 to 1989 at the former Wismut uranium mining company in Eastern Germany. By the end of 2003, a total of 3,016 lung cancer deaths, 3,355 deaths from extrapulmonary cancers, 5,141 deaths from heart diseases and 1,742 deaths from cerebrovascular diseases were observed. Although a number of studies have already been published on various endpoints in the Wismut cohort, the aim of the present analyses is to provide a direct comparison of the magnitude of radon-related risk for different cancer sites and cardiovascular diseases using the same data set, the same follow-up period and the same statistical methods. A specific focus on a group of cancers of the extrathoracic airways is also made here, due to the assumed high organ doses from absorbed radon progeny. Internal Poisson regression was used to estimate the excess relative risk (ERR) per unit of cumulative exposure to radon in working level months (WLM) and its 95% confidence limits (CI). There was a statistically significant increase in the risk of lung cancer with increasing radon exposure (ERR/WLM = 0.19%; 95% CI: 0.17%; 0.22%). A smaller, but also statistically significant excess was found for cancers of the extrathoracic airways and trachea (ERR/WLM = 0.062%; 95% CI: 0.002%; 0.121%). Most of the remaining nonrespiratory cancer sites showed a positive relationship with increasing radon exposure, which, however, did not reach statistical significance. No increase in risk was noted for coronary heart diseases (ERR/WLM = 0.0003%) and cerebrovascular diseases (ERR/WLM = 0.001%). The present data provide clear evidence of an increased radon-related risk of death from lung cancer, some evidence for an increased radon-related risk of death from cancers of the extrathoracic airways and some other extrapulmonary cancers, and no evidence for mortality from cardiovascular diseases. These findings are consistent with the results of other miner studies and dosimetric calculations for radon-related organ doses.     

Dietary fish oil supplemented with vitamin E improves quality indicators of rooster cold-stored semen through reducing lipid peroxidation

Cockerel semen is sensitive to cooling, which limits chilled storage of semen for more than 24 h. Results of artificial insemination with cold-stored semen are not desirable. This study was conducted to evaluate the effects of dietary fish oil and vitamin E (vitE) for cold-storage of rooster semen and its effects on parameters of semen during 48 h cooling preservation. Roosters were assigned into four dietary treatments; 1) control group received a basal diet, 2) vitE group received a basal diet supplemented with 200 mg/kg vitE, 3) fish oil group (FO) received a basal diet supplemented with 2% fish oil and 4) fish oil and vitE group received a basal diet supplemented with 2% fish oil and 200 mg/kg vitE (FO + vitE). Semen samples were collected after 40 days of feeding and then diluted and cooled to 5 °C for preservation up to 2 days. Several quality indicators of sperm such as motion characteristics, membrane integrity, and viability, and abnormal morphology, activity of mitochondria, lipid peroxidation and acrosome integrity of the sperm were assessed at different times of storage (0, 24 and 48 h). None of sperm were significantly affected by the diets at the start of storage (0 h, p > 0.05). FO and FO + vitE improved the percentage of total motility, viability, and mitochondria activity at 24 h (P ≤ 0.05). After 48 h, only FO + vitE group produced the higher percentage of total motility, viability and membrane integrity (P ≤ 0.05). Lipid peroxidation was significantly reduced in sperm obtained from roosters fed diets of FO + vitE and vitE compared to FO and control (P ≤ 0.05) at times of 24 and 48 h. There was no significant difference between control and vitE groups in none of parameters (P > 0.05). Integrity of acrosome and abnormal morphology were not significantly affected by the diets (P > 0.05). Supplementation of roosters' diet with 2% fish oil and 200 mg/kg vitamin E improved the quality of cold-stored semen by supporting several indicators of sperm quality through reducing lipid peroxidation.