Supplementation of Beltsville extender with quercetin improves the quality of frozen-thawed rooster semen.

1. Excessive reactive oxygen species (ROS) production during the sperm freeze-thawing process leads to membrane lipid peroxidation, DNA damage, and motility loss.2. This study examined the effect of supplementation of Beltsville poultry semen extender with different concentrations of quercetin (and antioxidants) on the cryopreservation of rooster sperm.3. Semen samples were collected from six Ross broiler breeders via abdominal massage twice a week for 4 weeks (eight replicates), and were divided into five equal aliquots to be diluted in Beltsville extenders that contained different concentrations of quercetin: 0, 5, 10, 15, and 20 mM. Motility, membrane functionality, abnormal morphology, lipid peroxidation, mitochondrial activity, viability, apoptosis status, and fertility potential were assessed post thaw.4. The addition of 10 and 15 mM quercetin to the semen extender significantly increased the total motility, straight-line velocity (VSL), and sperm membrane functionality compared with the other groups (P ≤ 0.05). Moreover, 10 mM quercetin caused higher progressive motility (34.86 ± 3.80%), curvilinear velocity (VCL; 175.11 ± 3.20 µm/s), average path velocity (VAP; 44.35 ± 11.06 µm/s), viability (59.14 ± 1.36%), mitochondrial activity (80.14 ± 2.07%), lower abnormal morphology (19.21 ± 0.45%), and lower lipid peroxidation (2.7 ± 0.13 nmol/ml) compared with the other groups (P < 0.05). The rate of fertility and hatchability after artificial insemination was not affected by experimental groups.5. In conclusion, supplementation of Beltsville extender with 10 mM quercetin could be a suitable method to improve post-thawed rooster sperm quality resulting in better freeze/thaw characteristics.

Supplementation of chilling storage medium with glutathione protects rooster sperm quality.

This study was aimed to evaluate the effect of addition of reduced glutathione (GSH) to the extender on the rooster's semen quality parameters and fertility potential. Semen samples were diluted with Lake extender contained 0, 0.5, 1, 2, 4 and 8 mM GSH. Then, were chilled to 5 °C and stored for a period of 48 h. Sperm motion characteristics, viability, membrane integrity, lipid peroxidation, mitochondrial activity and fertility potential were evaluated. At the initiation of the experiment (0 h), GSH did not affect sperm parameters, while 2-4 mM GSH improved (P ≤ 0.05) quality indicators during storage periods. Moreover, the samples treated with 2-4 mM GSH have had a lower lipid peroxidation compared to other groups (P ≤ 0.05). Artificial insemination using the semen samples, which had been stored in groups treated with 2-4 mM GSH for a period of 24 h, led to greater (P ≤ 0.05) fertilizing potential compared to the control group.

Effect of Glutathione Supplementation to Semen Extender on Post-thawed Rooster Sperm Quality Indices Frozen After Different Equilibration Times.

BACKGROUND: Avian sperm is susceptible to lipid peroxidation, compromising their fertility. The semen antioxidant system protects sperm plasma membrane against reactive oxygen species. OBJECTIVE: The study evaluates the effect of glutathione (GSH) addition to semen extender during different equilibration times (ET) on rooster sperm cryopreservation. MATERIALS AND METHODS: Semen samples are weekly collected from 60-week-old broiler breeder roosters. Collected samples were pooled and divided to six equal parts and frozen according to a randomized design (2 × 3 factorial arrangement). Treatments included adding two levels of GSH [0 (GSH-0) or 1 (GSH-1) mM] to semen extender during three ET: 0 (ET-0), 4 (ET-4) or 8 (ET-8) hours. Post-thawed motility and velocity parameters, apoptotic like changes, plasma membrane functionality, and mitochondrial membrane potential (MMP) were evaluated. RESULTS: Post-thawed total motility is improved in the GSH-1 compared to the GSH-0 group (P<0.10). Total motility responded quadratically to increasing levels of ET such that the highest value is recorded at ET-0. Although progressive motility (PM) is not affected by GSH or ET, the highest PM is obtained in the GSH-1×ET-0 group (P<0.05). The VAP and STR is improved in the GSH-1 compared to GSH-0 group; however, VAP decreases quadratically, and STR decreases linearly as ET is advanced (P<0.05). The interactive effect of GSH by ET tends (P<0.08) to affect the wobble coefficient (WOB), such that the highest value is recorded in the GSH-1×ET-0 group. Within both GSH supplemented and control groups, the amplitude of lateral head displacement (ALH) is highest (P<0.05) in the ET-0 group. The percentage of live spermatozoa quadratically decreases and the percentage of dead sperm quadratically increases in response to graded levels of ET (P<0.01). The highest plasma membrane functionality is also noted in the GSH-1×ET-0 group (P<0.05). Mitochondrial membrane potential quadratically decreases in response to increasing levels of ET (P<0.05). CONCLUSION: Generally, GSH supplementation to rooster sperm extender has some beneficial effects on post-thawed sperm motion characteristics, but does not positively interact with ET.

The effect of zinc oxide on rooster semen cryopreservation.

1. Deleterious effects from the freeze-thawing process on post-thawed sperm quality attributes are main limiting factors in cryopreservation. The current study was conducted to determine the effect of semen extender containing zinc oxide (ZnO) on post-thaw rooster sperm quality indices.2. Semen samples from six, 60-week-old broiler breeder roosters were collected weekly during five successive weeks. The samples were mixed and divided into three equal parts and diluted with semen extender containing different levels of ZnO; 0 (ZnO-0), 1 (ZnO-1) or 2 (ZnO-2) µg/ml. After thawing, motility and velocity parameters, plasma membrane functionality, apoptotic like changes, mitochondrial membrane potential (MMP), and DNA fragmentation index (DFI) were evaluated.3. Results showed that the addition of ZnO in the extender quadratically affected (P < 0.01) total motility (TM), progressive motility (PM), and average path velocity (VAP) with the highest values were noted in the ZnO-1 group. Levels of ZnO quadratically affected percentages of live (P < 0.01), apoptotic (P < 0.03) and dead (P < 0.10) spermatozoa, where the highest percentage of live, and the lowest percentage of apoptotic or dead spermatozoa was for the ZnO-1 group. Although adding ZnO quadratically affected plasma membrane functionality and MMP (P < 0.01), it did not affect (P > 0.05) DFI.4. In conclusion, there were some beneficial effects of ZnO supplementation in semen extender on post-thawed rooster sperm quality which may result in a better freezability.

Improvement of rooster semen quality using coenzyme Q10 during cooling storage in the Lake extender.

Sensitivity of rooster semen to stressful condition of cooling restricts the semen storage in commercial flocks for artificial insemination. This study was accomplished to investigate the effect of coenzyme Q10 (CoQ10) addition to the Lake extender during chilled-storage on the parameters of sperm quality and fertility performance. Roosters' pooled semen samples were assigned into equal parts and diluted with Lake extender supplemented with different concentrations of CoQ10 (0, 1, 2, 5 and 10 µM CoQ10). Then, semen samples were cooled to 5 °C and stored over 48 h. Total and progressive motilities, abnormal morphology, viability, membrane functionality, lipid peroxidation (LPO) and mitochondria active potential of diluted sperm were evaluated at 0, 24 and 48 h of cooling storage. Fertility performance of cooled stored semen was examined at 24 h of cooling storage. Although CoQ10 did not affect sperm quality at the starting time of cooling storage (0 h), extender supplementation with 5 µM of CoQ10 showed higher (P ≤ 0.05) sperm total and progressive motilities, membrane functionality, viability and mitochondria active potential at 24 h as well as total motility, viability and membrane functionality at 48 h in contrast with other groups. Moreover, lipid peroxidation was lower (P ≤ 0.05) in semen samples diluted with 5 µM CoQ10 at 24 and 48 h compared to others. After artificial insemination with 24 h chilled-stored sperm, fertility efficiency was higher (P ≤ 0.05) in treatments contained 5 µM CoQ10 compared to the control group. According to the results, using optimum dose of CoQ10 could be helpful to save rooster semen against chilled storage structural and functional damages.

L-carnitine is a survival factor for chilled storage of rooster semen for a long time.

Rooster sperm is sensitive to cooling, which restricts procedures to store sperms for extended periods of time for artificial insemination of commercial flocks. This study was conducted to evaluate the suitability of adding L-carnitine (LC) to chilled-storage of rooster sperm and its effects on sperm quality parameters and its fertility potential during storage at 5 °C. Pooled semen from roosters were divided into six equal aliquots and diluted with media supplemented with different concentrations of LC (0, 0.5, 1, 2, 4 and 8 mM LC). Diluted semen samples were cooled to 5 °C and stored over 48 h. Motility, viability, membrane functionality, lipid peroxidation and mitochondria activity of the sperm were assessed at 0, 24 and 48 h of storage. Moreover, fertility potential of chilled stored sperm was considered at 24 h of storage. While sperm quality was not affected by LC at the beginning of storage (0 h), supplementation of extender with 1 and 2 mM of LC significantly improved the percentage of sperm motility, viability, membrane integrity and mitochondria activity at 24 h and 48 h compared to other groups. Lipid peroxidation was significantly reduced in sperm samples diluted with 1 and 2 mM LC at 24 h (2.15 ± 0.52 nmol/ml and 2.21 ± 0.52 nmol/ml) and 48 h (3.42 ± 0.49 nmol/ml and 3.38 ± 0.49 nmol/ml) compared to other groups. Furthermore, fertility rates during artificial insemination using sperms cooled for 24 h in the presence of 1 and 2 mM LC were significantly higher (78%) than in the control group (64%). These findings suggest that optimum doses of LC could protect rooster sperm against cool storage-induced functional and structural damages.

L-Carnitine in rooster semen cryopreservation: Flow cytometric, biochemical and motion findings for frozen-thawed sperm.

Rooster semen cryopreservation is not efficient for artificial insemination in breeder flocks. L-Carnitine (LC) has been evaluated for effectiveness in cryopreservation media on the characteristics of rooster sperm after freeze-thawing. Motility characteristics, membrane functionality, abnormal morphology, apoptotic like changes, mitochondria activity and lipid peroxidation of rooster sperms were assessed after freeze-thawing with different concentrations of LC in Beltsville medium. Semen samples were collected from 12 roosters, twice a week, and diluted in the extenders that contained different concentrations of LC. Supplementation of Beltsevile with 1 and 2 mM LC was found to result in higher total motility (68.2± 1.7% and 69.1± 1.7%, respectively), progressive motility (28.4± 1.6%, 29.8± 1.6%), membrane functionality (76.2± 1.9% and 75.9± 1.9%), viability (58.2 ± 1.1%, 59.1 ± 1.1%) and lower significant of lipid peroxidation (2.53 ± 0.08 nmol/ml, 2.49 ± 0.08 nmol/ml) compared to control group containing no LC. Lower motility, progressive motility, and viability were observed in frozen-thawed sperm in extender containing 8 mM LC (35.8± 1.7%, 9.6± 1.2% and 27.1 ± 1.2%, respectively) compared to control. Morphology and mitochondrial activity were not affected by different concentrations of LC. Our results showed that supplementation of Beltsville extender with 1 and 2 mM LC significantly improved the quality of rooster sperm quality after freeze-thawing.

Orally administered Chrysin improves post-thawed sperm quality and fertility of rooster.

Chrysin is a bioflavonoid compound found in passion flower, chamomile, propolis and honey at high levels. Post-thawed sperm quality and fertility of Chrysin-fed roosters were assessed in this study. Twenty 40-week-old male broiler breeders were randomly divided into four groups and fed basal diet supplemented with different levels of Chrysin including 0 (Ch-0), 25 (Ch-25), 50 (Ch-50) or 75 (Ch-75) mg/day for 12 consecutive weeks. Semen samples were weekly collected from 6th to 9th week of experiment to evaluate some sperm quality parameters including total and progressive motility, plasma membrane integrity and functionality (in fresh and post-thawed samples) and mitochondrial activity (only in post-thawed samples). Also, collected semen samples from 10th, 11th and 12th week of experiment were frozen and then artificially inseminated to test fertility rate. According to the results, an improvement in both fresh and post-thawed sperm quality including total [fresh: 88.00 ± 0.58 and 87.25 ± 0.67 (p < .01); post-thawed: 51.07 ± 2.05 and 52.72 ± 1.96 (p < .01)] and progressive motility [fresh: 76.00 ± 0.58 and 78.25 ± 0.65 (p < .01); post-thawed: 40.61 ± 2.01 and 39.88 ± 2.01 (p < .01)], plasma membrane integrity [fresh: 91.60 ± 0.58 and 89.85 ± 0.59 (p < .01); post-thawed: 56.99 ± 1.86 and 54.39 ± 1.86 (p < .01)] and functionality [fresh: 75.40 ± 0.42 and 77.90 ± 0.96 (p < .01); post-thawed: 45.69 ± 1.71 and 46.35 ± 1.71 (p < .01)] was noted for both Ch-50 and Ch-75, respectively, groups compared to control group. Despite no significant change in mitochondrial activity, fertility rate of post-thawed spermatozoa was significantly improved in all Chrysin-fed groups compared to Ch-0 group. In conclusion, oral Chrysin administration to roosters could ameliorate cryopreservation-induced impairment of sperm quality and fertility rate.

Effect of tert-butyl hydroquinone on bull semen cryopreservation.

BACKGROUND: Many studies have been shown that freezing induced oxidative stress has detrimental effect on post-thaw sperm quality. OBJECTIVE: This study was conducted to investigate the effect of tert-butyl hydroquinone (tBHQ) on bull semen crtopreservation. MATERIALS AND METHODS: In this study, four different levels of tBHQ [Optidyl containing zero (T0), 2.5 (T2.5), 5 (T5), and 7.5 µM (T7.5) tBHQ] was used to study the effect of tBHQ on freezability of bull semen. On each collection day, four ejaculates were collected (a total of 24 ejaculates from four bulls), pooled and divided to four equal parts. Each part was diluted with one of the above-mentioned extenders and frozen. After thawing, sperm motility, plasma membrane functionality and integrity, apoptosis status and mitochondrial activity were assessed. RESULTS: The results show that total sperm motility was significantly higher in T5 compared to other groups. The value of VSL was significantly lower in T5 compared to T0. Also, T5 resulted in lower LIN and STR versus T0 and T2.5 groups. All extenders containing tBHQ resulted in a significantly higher percentage of sperm with functional membrane compared to T0 groups. Finally, Apoptosis related parameters and mitochondrial activity were not significantly difference between the groups. CONCLUSION: adding 5 µM tBHQ to the bull semen extender can be beneficial for post-thaw sperm quality. Also, in vivo or in vitro fertility test is recommended to test fertilizing ability of tBHQ exposed sperm.

The impact of using different doses of progesterone on memory performance.

OBJECTIVE: Progesterone is a sex hormone and its receptors are expressed throughout the hippocampus. This study was aimed at evaluating the effects of different doses of progesterone on memory. METHODS: Male rats were arbitrarily assigned to nine groups, namely Group I: control, Group II: control-cannula, Group III received 0.5 µl of saline by cannula, Groups IV , V, VI, VII and VIII received progesterone in doses of 0.5, 1, 1.5, 2, and 3 µg/ 0.5 µl by cannula, respectively. Group IX received 0.5 µl almond oil by cannula. Memory performance was tested in form of passive avoidance task. RESULTS: Our results indicated that progesterone at doses of 1.5 and 2 µg (p < 0.05) significantly increased the memory performance while at a dose of 3 µg (p < 0.05), it significantly decreased memory as compared to the control group. CONCLUSION: The current study revealed that the influence of progesterone on memory is related to its dose (Fig. 1, Ref. 25).

Fertility and flow cytometry study of frozen-thawed sperm in cryopreservation medium supplemented with soybean lecithin.

Semen cryopreservation can provide genetic resources for a large number of females from a small number of superior males. Optimization of cryopreservation media to achieve the highest quality of post-thaw semen is crucial. Soybean lecithin has evaluated as a plant-based cryoprotectant for substitution of egg yolk in ram semen extender. Flow cytometric and fertility assessments were applied following cryopreservation procedure in two experimental groups (SL group: extender containing 1% w/v soybean lecithin and EY group: extender containing 20% v/v egg yolk). The higher percentage of live sperm and the lower percentage of dead sperm were obtained in SL (47.66 ± 1.38, 52.33 ± 1.69, respectively) extender compared to EY (41.16 ± 1.38, 58.83 ± 1.69). For motion characteristics, plasma membrane integrity, acrosome integrity and mitochondria activity, no significant difference was observed between SL and EY extenders. In artificial insemination experiment, there was no significant difference in pregnancy rate, lambing rate and twining rate between SL and EY extenders. It can be concluded that SL extender can be an efficient alternative extender to preserve ram sperm during cryopreservation procedure without adverse effects.

Cryopreservation of ram semen in extenders containing soybean lecithin as cryoprotectant and hyaluronic acid as antioxidant.

A soybean lecithin-based extender supplemented with hyaluronic acid (HA) was assayed for effectiveness to improve the quality of frozen-thawed ram semen. HA has not been tested yet in an extender containing soybean lecithin for freezing ram semen. Thus, the aim of this study was to analyse the effects of soybean lecithin at 1% or 1.5% along with HA at 0, 0.5 and 1 mg ml(-1) in a Tris-based extender on the motion characteristics, membrane integrity (HOST), viability, GSH peroxidase (GSH-PX) activity, lipid peroxidation and acrosomal status after freezing-thawing. Semen was collected from four Mehraban rams during the breeding season and frozen in the six lecithin×HA extenders. The extender containing 1.5% lecithin supplemented with no HA yielded higher total motility (52.5%±1.6), viability (55.8%±1.6) and membrane integrity (44.5%±1.7), but the effects of the lecithin concentration did not reach signification. Linearity-related parameters, ALH, BCF, lipid peroxidation, GSH-PX activity, morphology and acrosomal status were not affected by the extender composition. In general, adding HA significantly decreased sperm velocity (1 mg ml(-1) HA), total motility (only with 1.5% lecithin), viability (1 mg ml(-1) HA for 1% lecithin; both concentrations for 1.5% lecithin) and membrane integrity. In conclusion, adding HA to the freezing extender supplemented with soybean lecithin failed to improve quality-related variables in ram semen. Increasing the lecithin content could have a positive effect, but further studies are needed.

Preservation of rooster post-thawed sperm epigenetic modifications, fertility potential and other quality parameters in different extenders using reduced glutathione.

Although rooster semen cryopreservation is an efficient procedure to spread qualified semen samples for reproductive goals, some post-thawed qualified semen samples resulted in poor fertility rate that could be related to epigenetic modifications during the cryopreservation process. This research was conducted to investigate the effect of reduced glutathione (GSH) in different cryopreservation extenders (Lake and Beltsville) on preservation of epigenetic modifications, fertility potential and other quality parameters of rooster sperm after thawing. Semen samples were collected and diluted in Lake and Beltsville extenders as follows: L-0: Lake without GSH, L-G: Lake with GSH, B-0: Beltsville without GSH, and B-G: Beltsville with GSH. After freeze-thawing process, sperm motility, membrane functionality, mitochondrial activity, acrosome integrity, viability, apoptosis status, lipid peroxidation, DNA fragmentation, ROS concentration, epigenetic modifications and fertility potential were evaluated. In results, the type of extender had no effect (P > 0.05) of post-thawed sperm quality. The treatments containing GSH presented higher (P ≤ 0.05) total motility, progressive motility, membrane functionality, mitochondrial activity, acrosome integrity, viability, DNA methylation, fertility as well as lower (P ≤ 0.05) lipid peroxidation, apoptosis, DNA fragmentation and ROS concentration than other treatments. Extender supplementation with GSH had no effect (P > 0.05) on histone methylation, histone acetylation and hatching rate. In conclusion, supplementation of rooster sperm cryopreservation extender with GSH could be an effective strategy to preserve post-thawed sperm DNA methylation, fertility and other quality parameters during reproductive programs.

Optimization of ram semen cryopreservation using a chemically defined soybean lecithin-based extender.

The purpose of the present study was to investigate the effects of a chemically defined soybean lecithin-based semen extender as a substitute for egg yolk-based extenders in ram semen cryopreservation. In this study, 28 ejaculates were collected from four Zandi rams in the breeding season and then pooled together. The pooled semen was divided into six equal aliquots and diluted with six different extenders: (i) Tris-based extender (TE) containing 0.5% (w/v) soybean lecithin (SL0.5), (ii) TE containing 1% (w/v) soybean lecithin (SL1), (iii) TE containing 1.5% (w/v) soybean lecithin (SL1.5), (iv) TE containing 2% (w/v) soybean lecithin (SL2), (v) TE containing 2.5% (w/v) soybean lecithin (SL2.5) and (vi) TE containing 20% (v/v) egg yolk (EYT). After thawing, sperm motility and motion parameters, plasma membrane and acrosome integrity, apoptosis status and mitochondrial activity were evaluated. The results shown that total and progressive motility (54.43 ± 1.33% and 25.43 ± 0.96%, respectively) were significantly higher in SL1.5 when compared to other semen extenders. Sperm motion parameters (VAP, VSL, VCL, ALH and STR) were significantly higher in SL1.5 compared to other extender, with the exception of SL1 extender. Plasma membrane integrity (48.86 ± 1.38%) was significantly higher in SL1.5 when compared to other semen extenders. Also, percentage of spermatozoa with intact acrosome in SL1.5 (85.35 ± 2.19%) extender was significantly higher than that in SL0.5, SL2.5 and EYT extenders. The results showed that the proportion of live post-thawed sperm was significantly increased in SL1.5 extender compared to SL0.5, SL2 and EYT extenders. In addition, SL1, SL1.5 and SL2.5 extenders resulted in significantly lower percentage of early-apoptotic sperm than that in EYT extender. There were no significant differences in different semen extenders for percentage of post-thawed necrotic and late-apoptotic spermatozoa. Also, the results indicated that there are slight differences for percentage of live spermatozoa with active mitochondria between extenders. In conclusion, SL1.5 extender was better than other extenders in most in vitro evaluated sperm parameters.

Cysteamine enhances quality and fertility potential of rooster semen in cooled storage.

This study investigated the effects of supplementing Lake extender with cysteamine (CYS) on rooster semen quality in cold storage and it's fertility performance. Semen samples were diluted with Lake extender supplemented with different concentrations of CYS (0, 1, 2, 4 and 8 mM) and were cooled and stored at 5 °C for a period of 46 h. Motility, membrane functionality, viability, lipid peroxidation, and mitochondria membrane potential were evaluated at 0, 23 and 46 h of storage. Fertility was assessed at 23 h of storage. Although at the beginning time (0 h), parameters were not affected, 1 mM of CYS improved (P ≤ 0.05) total motility, progressive motility and mitochondria membrane potential during 23 and 46 h storage. Moreover, 1 and 2 mM CYS improved (P ≤ 0.05) membrane functionality and viability compared to other groups. Lipid peroxidation was lower (P ≤ 0.05) in samples diluted with 1 and 2 mM CYS compared to the others. Artificial insemination with 23-hrs cooled-stored semen produced the higher (P ≤ 0.05) fertility rate in groups received 1 and 2 mM CYS compared to the control group. In conclusion, addition of 1 and 2 mM CYS to the extender could be helpful to protect rooster semen against structural and functional damages of cooling storage process.

Is leaf dry matter content a better predictor of soil fertility than specific leaf area?

BACKGROUND AND AIMS: Specific leaf area (SLA), a key element of the 'worldwide leaf economics spectrum', is the preferred 'soft' plant trait for assessing soil fertility. SLA is a function of leaf dry matter content (LDMC) and leaf thickness (LT). The first, LDMC, defines leaf construction costs and can be used instead of SLA. However, LT identifies shade at its lowest extreme and succulence at its highest, and is not related to soil fertility. Why then is SLA more frequently used as a predictor of soil fertility than LDMC? METHODS: SLA, LDMC and LT were measured and leaf density (LD) estimated for almost 2000 species, and the capacity of LD to predict LDMC was examined, as was the relative contribution of LDMC and LT to the expression of SLA. Subsequently, the relationships between SLA, LDMC and LT with respect to soil fertility and shade were described. KEY RESULTS: Although LD is strongly related to LDMC, and LDMC and LT each contribute equally to the expression of SLA, the exact relationships differ between ecological groupings. LDMC predicts leaf nitrogen content and soil fertility but, because LT primarily varies with light intensity, SLA increases in response to both increased shade and increased fertility. CONCLUSIONS: Gradients of soil fertility are frequently also gradients of biomass accumulation with reduced irradiance lower in the canopy. Therefore, SLA, which includes both fertility and shade components, may often discriminate better between communities or treatments than LDMC. However, LDMC should always be the preferred trait for assessing gradients of soil fertility uncoupled from shade. Nevertheless, because leaves multitask, individual leaf traits do not necessarily exhibit exact functional equivalence between species. In consequence, rather than using a single stand-alone predictor, multivariate analyses using several leaf traits is recommended.

Rooster frozen-thawed semen quality following sublethal xanthine oxidase treatments.

Reactive oxygen species are associated with cryodamage and may be a factor causing or exacerbating cellular cryodamage during freezing and thawing processes. Induction of sublethal oxidative stress as a new approach for preconditioning of sperm improves the cryo-resistance of sperm. The aim of this study was to investigate effects of sublethal concentrations of xanthine oxidase (XO), which induces oxidative stress before cryopreservation on values for semen quality variables of rooster sperm post-thawing. Semen samples were collected from 15 roosters and treated with different concentrations of XO [XO-0, XO-0.005, XO-0.05, XO-0.5, XO-5, and XO-50 U/ml]; then, the effects of treatments with XO as sublethal stressors, were examined. Results indicated the XO-0.5 and XO-5 treatments resulted in a greater percentage of sperm total motility, progressive motility, viability, and membrane functionality compared to other groups. There was no difference after treatments with XO-0, XO-0.005, and XO-0.05 on sperm total motility, membrane functionality, apoptosis, mitochondria activity, and viability. There was a greater percentage of mitochondria activity in sperm of the XO-0.05, XO-0.5, and XO-5 groups. Furthermore, there was the greatest concentration of malondialdehyde (MDA) in samples of the XO-50 group. Values for sperm abnormal morphology, acrosome integrity, and DNA fragmentation were not different among samples post-thawing. Sperm treated with XO-0.5 and XO-5 had a greater fertilization capacity than those of the control group. In conclusion, treatment of sperm with 0.5 and 5 U/ml XO as inducers of mild oxidative stress before cryopreservation, improved several function quality indices of sperm post-thawing.

Stomatal vs. genome size in angiosperms: the somatic tail wagging the genomic dog?

BACKGROUND AND AIMS: Genome size is a function, and the product, of cell volume. As such it is contingent on ecological circumstance. The nature of 'this ecological circumstance' is, however, hotly debated. Here, we investigate for angiosperms whether stomatal size may be this 'missing link': the primary determinant of genome size. Stomata are crucial for photosynthesis and their size affects functional efficiency. METHODS: Stomatal and leaf characteristics were measured for 1442 species from Argentina, Iran, Spain and the UK and, using PCA, some emergent ecological and taxonomic patterns identified. Subsequently, an assessment of the relationship between genome-size values obtained from the Plant DNA C-values database and measurements of stomatal size was carried out. KEY RESULTS: Stomatal size is an ecologically important attribute. It varies with life-history (woody species < herbaceous species < vernal geophytes) and contributes to ecologically and physiologically important axes of leaf specialization. Moreover, it is positively correlated with genome size across a wide range of major taxa. CONCLUSIONS: Stomatal size predicts genome size within angiosperms. Correlation is not, however, proof of causality and here our interpretation is hampered by unexpected deficiencies in the scientific literature. Firstly, there are discrepancies between our own observations and established ideas about the ecological significance of stomatal size; very large stomata, theoretically facilitating photosynthesis in deep shade, were, in this study (and in other studies), primarily associated with vernal geophytes of unshaded habitats. Secondly, the lower size limit at which stomata can function efficiently, and the ecological circumstances under which these minute stomata might occur, have not been satisfactorally resolved. Thus, our hypothesis, that the optimization of stomatal size for functional efficiency is a major ecological determinant of genome size, remains unproven.

The mitochondria-targeted antioxidant Mito-TEMPO conserves rooster's cooled semen quality and fertility potential.

The PUFAs content of rooster sperm cells makes them vulnerable to the thermal shocks during chilling storage, which reduces the fertility performance of cooled sperm. Extender supplementation with antioxidants is a reasonable method to conserve sperm fertility potential during cooling storage process. The aim of this study was to determine the effect of Mito-TEMPO addition to the Lake medium on rooster sperm quality and fertility potential during cooling process. Semen samples were diluted in the Lake medium and assigned into five equal aliquots and supplemented with 0, 0.5, 5, 50 and 500 µM Mito-TEMPO. Then, the samples were cooled at 5 °C and conserved up to 50 h. Total motility, progressive motility, morphology, viability, membrane integrity, lipid peroxidation and mitochondrial activity of samples were analyzed during 0, 25 and 50 h of cooling period. Artificial insemination was also conducted using 25 h-cooled semen. No significant difference was observed among different treatments during quality evaluations at 0 h storage. Extender supplementation with 5 and 50 µM Mito-TEMPO presented greater (P ≤ 0.05) total motility, progressive motility, viability, membrane integrity and lower lipid peroxidation compared to other groups during 25 and 50 h cooling storage. Mitochondrial activity was higher (P ≤ 0.05) in groups received 5, 50 and 500 µM Mito-TEMPO than others. Fertility rate of 25 h-cooled-stored samples was higher (P ≤ 0.05) in groups containing 5 and 50 µM Mito-TEMPO compared to control group. In conclusion, addition of 5 and 50 µM Mito-TEMPO as a mitochondria-targeted antioxidant to the storage medium could be a suitable method to conserve rooster semen quality against stressful conditions of cooling storage process.

Effects of reduced glutathione on the quality of rooster sperm during cryopreservation.

The purpose of this study was to investigate the beneficial effects of reduced glutathione (GSH) for cryopreservation of rooster semen. In experiment 1, semen samples were collected from 15 roosters and diluted in the Lake extender that contained various concentrations of GSH as follows: Lake without GSH (control, GSH 0), Lake containing 0.5 mM (GSH 0.5), 1 mM (GSH 1), 2 mM (GSH 2), 4 mM (GSH 4) and 8 mM (GSH 8) GSH. Viability, membrane functionality, morphology, mitochondrial activity, acrosome integrity, motion parameters, lipid peroxidation and DNA fragmentation were assessed after thawing. In experiment 2, reproductive performance of thawed semen was evaluated via artificial insemination. Supplemented extenders with 2 and 4 mM GSH presented higher (P ≤ 0.05) viability (59.4 ±â€¯2.4% and 60.8 ±â€¯2.4%), membrane functionality (62.3 ±â€¯2.6% and 64.7 ±â€¯2.6%), mitochondrial activity (49.4 ±â€¯1.7% and 49.8 ±â€¯1.7%), total motility (57.1 ±â€¯1.9% and 58.8 ±â€¯1.9%, respectively), progressive motility (28.9 ±â€¯1.3% and 29.6 ±â€¯1.3%), and lower lipid peroxidation (2.4 ±â€¯0.09 nmol/ml and 2.3 ±â€¯0.09 nmol/ml) compared to control group. Acrosome integrity was higher (P ≤ 0.05) in GSH 4 (91.4 ±â€¯1.8%) compared to other groups. DNA fragmentation and MDA concentrations were higher (P ≤ 0.05) in GSH 8 (12 ±â€¯1.2% and 3.4 ±â€¯0.09 nmol/ml). In experiment 2, higher (P ≤ 0.05) fertility rate was observed in GSH 2 and GSH 4 (61.9% and 63.8%, respectively) compared to control (41.4%) group. In conclusion, supplementation of Lake extender with 2 and 4 mM GSH improves the cryo-survival and fertility potential of rooster sperm and it could be an applied method for improvement of reproductive goals.