Quercetin supplemented casein-based extender improves the post-thaw quality of rooster semen

The advantageous influence of quercetin (Q) supplementation in an extender has not yet been evaluated for rooster semen cryopreservation. This research was purposely conducted in order to assess the effect of different quercetin concentrations added into an extender on the sperm quality of the rooster subsequent to a freezing-thawing process. After the freezing-thawing process, spermatozoa quality parameters (membrane functionality, acrosome integrity, motility, viability, and abnormal morphology), endogenous enzymes (SOD, CAT, and GPx), mitochondrial activity, DNA fragmentation index, lipid peroxidation (MDA), and ROS were all evaluated. A total of 75 neat pooled ejaculates (3 ejaculates/rooster) were collected from 25 arbor acres roosters (24 wks) twice a week using abdominal massage technique, then divided into five equal aliquots and diluted with an extender containing different doses of Q (CS-Q) as follows: casein extender without Q (control only), casein extender containing 0.040 mg/mL quercetin (CS-Q 0.040), 0.020 mg/mL quercetin (CS-Q 0.020), 0.010 mg/mL quercetin (CS-Q 0.010), and 0.005 mg/mL quercetin (CS-Q 0.005). Our results depicted that adding to the extender with a 0.010 mg/mL Q enhanced (P < 0.01) sperm motility, membrane function, viability, mitochondrial activity, intact acrosome (P < 0.05), SOD (P < 0.001), CAT, and GPx (P < 0.01) compared to the control group at post-thaw. Compared to the control group and other treatment groups after the freeze-thawing process, the addition of 0.005 mg/mL Q into the extender also showed higher (P < 0.05) improvement in the quality of sperm parameters and a higher (P < 0.01) SOD and CAT but did not improve mitochondrial activity and sperm viability. In addition, there was a lower degree of DNA fragmentation index, lower (P < 0.05) lipid peroxidation and ROS in frozen-thawed sperm treated with 0.010 mg/mL and 0.005 mg/mL Q than in control and the other treatment groups. In addition, 0.020 mg/mL Q supplementation into the extender also reduced DNA fragmentation and improved GPx activity compared to the control group at post-thaw. Different concentrations of Q 0.010 and 0.005 mg/mL added to the extender reduced the percentage of abnormal spermatozoa compared to the other groups. The results of this study showed for the first time that the inclusion of an extender with a suitable quercetin concentration of 0.010 mg/mL improved the post-thawed quality of rooster semen.     

Idebenone improves quality of ram sperm by mitigating oxidative stress during cryopreservation

The present study was designed to test the effect of different levels of idebenone, a potent antioxidant on the quality of ram semen at post thaw. Eighteen (18) ejaculates were collected and extended with tris extender supplemented with no antioxidant (CON), with 2 μM idebenone (Id2), 5 μM idebenone (Id5), 7.5 μM idebenone (Id7.5) and 10 μM idebenone (Id10). The sperm quality was determined in terms of percent sperm motility, live sperm percentage, percent hypoosmotic swelling test (HOST) positive spermatozoa and percent intact acrosome (PIA). Moreover, malondialdehyde (MDA) level, an end product of lipid peroxidation (LPO) was also measured at post thaw both in seminal plasma and sperm cell. At post thaw, the percent sperm motility was significantly higher (p < 0.05) for Id10 as compared to Id2, Id5, Id7.5 and control. The live sperm percentage was non-significantly (p > 0.05) higher for Id10 as compared to control, Id5 and Id7.5 but significantly higher than Id2. The percent HOST positive spermatozoa was significantly higher (p < 0.05) for Id10 than control, Id2 and Id5. The MDA level in seminal plasma was significantly lower (p < 0.05) for Id10 than control and Id2. The MDA level in spermatozoa did show similar trend as in seminal plasma. Further, all the sperm parameters at all idebenone levels declined significantly from pre freeze to post thaw. In conclusion, idebenone at 10 μM level improved post thaw sperm quality by mitigating peroxidative stress, hence could be considered as a promising antioxidant additive for cryopreservation of ram semen.     

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 trace elements on the seminal oxidative status and correlation to sperm motility in infertile Saudi males

The impact of trace elements, especially zinc, selenium, copper, and magnesium, on male fertility has gained great interest and significance. Increased oxidative stress and altered trace element levels are probable etiological factors underlying male reproductive dysfunction and infertility. The present study focused on the evaluation of seminal oxidative markers, such as reactive oxygen species (ROS), malondialdehyde (MDA), and total antioxidant capacity (TAC), and trace element levels in the normozoospermic fertile control group (n = 40) and asthenozoospermic infertile group (n = 30). Semen from infertile men exhibited significantly higher ROS and MDA levels accompanied with significant decline in TAC and trace element (zinc and magnesium) levels. Furthermore, a significant correlation was observed between trace elements and oxidative markers with sperm motility. The current study revealed increased lipid peroxidation and oxidant-reductant imbalance that leads to deterioration of semen quality and male infertility. Thus, oxidative stress and trace elements can be considered important biomarkers of male infertility. Measurement of seminal oxidative stress with conventional seminological parameters must be integrated in fertility assessment from early stages to ensure healthy semen characteristics and fertility in men.     

The influence of macro and trace elements on sperm quality

The aim of this study was to examine the association between combined concentrations of macro and trace elements and markers of oxidative stress and antioxidative defense system function together with selected cytokine levels. Based on the combined medians of the seminal plasma levels of calcium, magnesium, zinc, copper, iron, and selenium, the study subjects (88 fertile male volunteers) were divided into the following two subgroups: the Me-L group (low level of metals) and the Me-H group (high level of metals). There was a tendency toward reduced motility in the Me-H group compared to that in the Me-L group. The total protein, albumin, and total oxidation status (TOS) levels were significantly higher in the Me-H group than in the Me-L group. The total superoxide dismutase (SOD), Mn-SOD, and CuZn-SOD, activity in spermatozoa were significantly lower in the Me-H group than in the Me-L group. In seminal plasma, the Mn-SOD activity was significantly higher in the Me-H group, whereas the CuZn-SOD activity was significantly lower. Additionally, the activity levels of glutathione peroxidase (GPx) and glutathione-S-transferase (GST) were lower in the Me-H group. The medians of IL-1β, IL-10, and IL-12 were significantly higher in the Me-H group than in the Me-L group, whereas the medians of IL-2, IL-5, and IL-13 were significantly lower. Higher levels of macro and trace elements in the seminal plasma of fertile males may be associated with decreased motility. Higher levels of the examined metals are associated with elevated oxidative stress accompanied by decreased activities of some of the antioxidant enzymes and increased pro-inflammatory cytokine levels.     

The effects of antioxidants on semen traits and in vitro fertilizing ability of sperm from the flat-headed cat (Prionailurus planiceps)

Since antioxidants can overcome the negative effects of reactive oxygen species (ROS) during sperm cryopreservation, post-thaw sperm quality in flat-headed cats (Prionailurus planiceps), an endangered species, might benefit from the addition of antioxidants to semen extender. The objectives of this study were to: 1) investigate semen traits; and 2) evaluate effects of the vitamin E analogue Trolox (vitamin E) and glutathione peroxidase (GPx) on the quality of frozen sperm from captive flat-headed cats in Thailand. Eight ejaculates were collected by electroejaculation from four flat-headed cats. Each semen sample was divided into three aliquots and re-suspended in a semen extender as follows: 1) without antioxidant supplementation (control); 2) supplemented with 5 mM vitamin E; or 3) supplemented with 10 U/mL GPx. All samples were cryopreserved and thawed. Subjective sperm motility, progressive motility, and the integrity of the sperm membrane, acrosome and DNA were evaluated at semen collection, after 1 h cold storage, and at 0 and 6 h after thawing. Mitochondrial membrane potential, early apoptotic cells, and embryo development by heterologous in vitro fertilization were evaluated after thawing. Captive flat-headed cats were affected by teratozoospermia. After 1 h cold storage, sperm membrane integrity in samples supplemented with GPx was higher than the control group (54.5 ± 13.7 vs 51.3 ± 13.9; P < 0.05; mean ± SD). Sperm frozen in extender with GPx had higher motility at 6 h and greater mitochondrial membrane potential at 0 and 6 h post-thaw incubation than the other groups (P < 0.05). In conclusion, GPx improved the quality of frozen-thawed sperm in flat-headed cats.     

Effects of anti-oxidant additives on microscopic and oxidative parameters of Angora goat semen following the freeze–thawing process

The anti-oxidant system of reduced glutathione (GSH), glutathione peroxidase (GSH-PX), catalase (CAT), and superoxide dismutase (SOD) has been described as a defense functioning mechanism against lipid peroxidation (LPO) in semen, and is important in maintaining sperm motility and viability. This anti-oxidant capacity of sperm cells may be insufficient in preventing LPO during the freeze–thawing process. The aim of this study was thus to determine the influence of varying doses of anti-oxidant additives on standard semen parameters, lipid peroxidation and anti-oxidant activities after the freeze–thawing of goat semen. Ejaculate samples (artificial vagina) obtained from 4 mature Angora goats were evaluated and pooled at 37 °C. The semen samples diluted with a Tris-based extender, containing taurine (25, 50, 75 mM), trehalose (25, 50, 75 mM), and cysteine (5, 10, 15 mM), and an extender containing no anti-oxidant additives (control) were again evaluated. Diluted semen was cooled down to 5 °C and frozen in 0.25 ml French straws, prior to being stored in liquid nitrogen. Frozen straws were thawed in a water bath (37 °C) for 30 s for microscopic sperm evaluation. Upon evaluation of parameters for semen quality, the use of a Tris-based extender supplemented with anti-oxidant additives was found to cause no significant improvement in sperm mortality, when compared to the controls. Increasing doses of taurine and trehalose decreased (P < 0.05) the sperm motility following the freeze–thawing of the goat semen. In biochemical assays, the application of taurine (75 mM) produced the lowest level of malondialdehyde (MDA) (4.46 ± 0.31 nmol/ml), compared to the controls (P < 0.001). Lower GSH levels were higher in the groups in which cysteine was included at 10 and 15 mM (3.27 ± 0.11 and 3.45 ± 0.28 nmol/ml) – compared to the group which received 5 mM cysteine, as well as the controls (2.27 ± 0.08 and 2.50 ± 0.08 nmol/ml respectively, P < 0.001). Compared to the controls, taurine at a concentration of 25 and 75 mM, and increasing doses (50 and 75 mM) of trehalose, significantly increased the GSH-PX activity (P < 0.01). The maintenance of CAT activity was demonstrated to be higher with the addition of 10 and 15 mM cysteine, compared to the other groups (P < 0.001). Vitamin A (VitA) levels were significantly higher, compared to the controls (267.34 ± 9.68 mg/dl and 267.34 ± 9.68 mg/dl, respectively), when 25 mM taurine (329.61 ± 6.35 mg/dl) and 10 mM (318.64 ± 6.34 mg/dl) cysteine was added to the extender (P < 0.001). The results of this study provide a new approach to the cryopreservation of Angora goat semen and could contribute to the improvement of this technology in the goat industry.     

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.     

Effect of daily semen centrifugation and resuspension on the longevity of equine sperm quality following cooled storage

An experiment was conducted to determine whether cooled semen quality could be maintained for a longer interval by conducting daily centrifugation of extended semen, with resuspension of the sperm pellet in fresh extender. Semen treatments included SP10NC and SP50NC which contained 10 and 50% seminal plasma, respectively, were not centrifuged (NC), and were stored at 4 to 7 °C for 96 h. Treatments SP10C and SP50C contained 10 and 50% seminal plasma, respectively, but were centrifuged (C) after 24, 48, and 72 h of cooled storage, with daily resuspension in fresh extender containing 10% seminal plasma. Percent total sperm motility (TMOT) and progressively motile (PMOT) was reduced (P < 0.05) in the SP50NC treatment after 24, 48, 72, and 96 h of storage, and TMOT did not differ (P > 0.05) in the SP10C, SP50C, SP10NC groups after the same storage periods. The % COMP-_αt did not differ (P > 0.05) among treatments at any time period. Percent membrane intact sperm (SMI) was reduced in SP50NC, as compared to SP10C at 48, 72, and 96 h (P < 0.05). Daily centrifugation and resuspension of sperm exposed to 50% seminal plasma for the first 24 h (SP50C) yielded similar TMOT, PMOT, VCL, SMI, % COMP-_αt (P > 0.05) to Groups SP10NC and SP10C after 96 h of storage. Daily centrifugation and resuspension of cool-stored equine semen in fresh extender may be a method to increase sperm longevity.     

Effect of age and environmental factors on semen quality, glutathione peroxidase activity and oxidative parameters in simmental bulls

Taking into account that semen quality depends on animal age and climate conditions and that oxidative stress has been reported to be a common cause of infertility, the objective of this study was to monitor indicators of oxidative stress and antioxidant protection during four seasonal periods in service bulls of various age to get better insight into the significance of these factors upon evaluating service bull semen. The research was conducted over a year on 19 Simmental service bulls. Animals were divided into two groups according to age; Group I consisted of younger bulls aged two to four yrs (n = 9), and Group II was comprised of older bulls aged five to ten yrs (n = 10). Semen samples were obtained once in the middle of every seasonal period and blood samples for biochemical analysis were collected by jugular venipuncture immediately after ejaculate collection. The activity of total glutathione peroxidase (T-GSH-Px), selenium-dependent glutathione peroxidase (Se-GSH-Px) and selenium-independent glutathione peroxidase (non-Se-GSH-Px), together with the intensity of lipid peroxidation (thiobarbituric acid reactive substances; TBARS) and oxidative protein damage (protein carbonyl content (PCC)) were measured in seminal plasma. In samples of spermatozoa and blood serum, the activity of Se-GSH-Px and TBARS and PCC concentrations were determined. Older service bulls had significantly higher ejaculate volume in summer in comparison with younger bulls, whereas the number of spermatozoa and progressive motility percentage did not significantly vary with age. Younger animals had lower progressive motility percentage during summer than in spring, with more intensive oxidative processes observed in seminal plasma (TBARS) and spermatozoa (TBARS and PCC). Based on the results presented here, it can be concluded that younger bulls are more sensitive to elevated ambient temperatures during the summer, when intensified prooxidative processes in semen plasma and spermatozoa eventually led to decreased sperm progressive motility with consequential semen quality deterioration.     

Effect of different concentrations of l-carnitine in extender for semen cryopreservation in sheep

This study assessed the effect of l-carnitine (LC) in sheep semen extenders containing or not egg yolk for cryopreservation in sheep. Two extenders (TRIS-egg yolk or the commercial optiXcell™ IMV medium) were used, totaling six groups: IMV – (0, 5 and 10 mM LC) and TRIS – (0, 5 and 10 mM LC). After the freezing-thawing process and throughout incubation at 38 °C for up to 3 h, several parameters were evaluated: sperm kinetics, hypoosmotic, plasma membrane integrity, capacitation status and lipid peroxidation level. The supplementation of either 5 or 10 mM LC randomly affected some parameters and, overall, TRIS was superior (P < 0.05) than IMV extender. In the LC-groups, IMV had greater (P < 0.05) oxidative stress than TRIS. In conclusion, although LC affected isolated parameters, its supplementation in semen extender had no consistently beneficial effect on freezing-thawing ram sperm.     

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.     

The behaviour of centrioles and the formation of the flagellum in rooster and drake spermatids

Summary Developmental changes in the formation of the centrioles and flagellum during spermiogenesis in the rooster and drake were studied.Changes in the length and thickness of the wall of the centrioles were observed from an early stage of spermatid development. Before the proximal centriole is attached to the nucleus microtubules were observed near the centrioles joined to them. At this stage of spermatid development changes on the nuclear membrane were observed at a place where the proximal centriole is attached to the nucleus. At the later stage of spermatid differentiation three to five dense extensions in the space of the nuclear invagination and dense bodies or granules near the distal centriole were present. The anterior part of the newly formed flagellum is covered by a cytoplasmic membrane displaying extension which is approximately 1.3 m long. Slight differences between the two species were observed.     

Effect of organic selenium on turkey semen quality during liquid storage

The aim of the present study was to evaluate the effect of dietary organic selenium on the turkey semen during storage. Twenty males (BUT, Big 6, 40 weeks of age) were divided into control (n = 10) and experimental group (n = 10). The turkeys in the both groups were fed with a commercial diet containing 0.1 ppm Se in the form of sodium selenite. The experimental birds were additionally supplied with 0.3 ppm organic Se in the form Sel-Plex™ (Alltech, Inc.). After 30 days of feeding, the semen samples were collected twice a week for the 3 weeks of the study and diluted 1 + 1 (v/v) with TUR-2 diluent, and stored in a water bath (+10 to 15° C) for 6 h. The percentage of motile spermatozoa, the sperm viability (live/dead spermatozoa), total lipids, phospholipids and total cholesterol were assessed in fresh and stored semen. The fertilizing ability of semen was assessed by artificial insemination of 30 hens per group with dose containing 200 × 10⁶ spermatozoa weekly. After 6 h of semen storage, the motility of spermatozoa decreased significantly in the control group (by 8.7 relative percent, P < 0.05) and only by four relative percent (P > 0.05) in experimental group reflecting a protective effect of dietary Se supplementation. The proportion of live spermatozoa was higher in fresh semen and significantly lower in stored semen. The positive effect of Se supplementation was observed on the lipid composition of stored semen: the concentration of the total lipids and phospholipids in the seminal plasma from control group significantly increased, while in the experimental group remained constant. Better semen integrity in the experimental group was associated with an improved fertilizing ability of spermatozoa: the fertility rate of stored spermatozoa in the control group was 88%, while in the experimental group was 90.5%.     

Effect of chronic cyclic heat stress on the intestinal morphology,oxidative status and cecal bacterial communities in broilers

The objective of this study was to examine the effects of chronic cyclic heat stress (HS) on the intestinal morphology, oxidative stress and cecal bacterial communities of broilers. One-day-old Arbor Acres (AA) male broilers (n = 100) were acclimated for 3 weeks and then randomly allocated into two groups, normal control (NC) group (22 ± 1 °C, 24 h/day) and HS group (32 ± 1 °C, 10 h/day lasted for 2 weeks). At 35 d of age, intestinal segments (duodenum, jejunum and ileum) and cecal digesta were collected for detection. HS affected intestinal morphology, inducing epithelial cell abscission, inflammatory cell infiltration, and lamina propria edema. Compared with the NC group, HS significantly decreased (P < 0.01) villus height (VH) and the VH-to-crypt depth (CD) ratio (VCR), increased (P < 0.05) CD in the duodenum and ileum, but had no effect on the VH in the jejunum. Moreover, HS induced oxidative stress with antioxidant enzymes activity decreasing (P < 0.05) while malondialdehyde (MDA) content increasing (P < 0.05) in small intestine. Pearson's correlation analysis indicated that MDA content was negatively correlated with VH (P < 0.05). The result of 16S rRNA sequencing showed that HS exposure impacted cecal microbiota alpha diversity (phylogenetic diversity whole-tree index) and beta diversity. Based on principal coordinate analysis (PCoA) plots for weighted UniFrac metrics and unweighted pair group method with arithmetic mean (UPGMA), there were 8 discriminative features at the genus level (linear discriminant analysis score > 2). Parabacteroides, Saccharimonas, Romboutsia and Weissella were reduced, while Anaerofustis, Pseudonocardia, Rikenella and Tyzzerella were enriched in heat-stressed broilers. Collectively, these results indicated that chronic cyclic HS induced oxidative stress that caused damage to intestinal villus-crypt structures, and then altered the cecal microflora profile.     

Caffeic acid improves microscopic sperm parameters and antioxidant status of buffalo (Bubalus bubalis) bull semen following freeze-thawing process

This study investigates the effects of cryopreservation and supplementation of buffalo's semen with Caffeic acid. It studies the effects of different Caffeic acid concentrations on cryopreservation capacity of the buffalo and evaluates their influence on various sperm parameters like motility, viability, progressive motility, sperm plasma membrane integrity, and antioxidant status. Twenty-four semen samples were collected with an artificial vaginal from three adult water buffalos. The semen samples were evaluated and the qualified ejaculates were separated and were diluted in a Tris-based extender. The resulting samples were classified into 5 groups: No antioxidant (control), Control sham (NaOH), Caffeic acid 50 μM, Caffeic acid 100 μM, and Caffeic acid 200 μM. The semen samples encountered cryodamage and the quality was deteriorating during the cryopreservation (P < 0.05). The semen evaluation after thawing showed that the groups of samples receiving 100 μM Caffeic acid had higher viability, total motility, and lower abnormal sperm and better linearity (LIN), curvilinear velocity (VCL), straight-line velocity (VSL) and path velocity and higher intact plasma membrane (P < 0.05) compared to other groups. It is notable that adding 100 μM Caffeic acid to freezing extenders enhances the CAT, GPx, SOD, and GSH and also ameliorates total antioxidant capacity of spermatozoa after thawing. It is notable that the addition of 100 μM Caffeic acid decreases the amount of Malondialdehyde. These reactions lead us to conclude that 100 μM Caffeic acid enhances the semen quality of water buffalo after thawing.     

Effect of low density lipoprotein on the quality of cryopreserved dog semen

Egg yolk is included in extenders for semen cryopreservation due to its protective effect against cold shock, which is attributed to the presence of low density lipoprotein (LDL). This study evaluates how semen quality is affected by using LDL as a replacement for egg yolk in extenders for cooled and frozen dog semen. In Experiment 1, semen was extended in TRIS–glucose at 5 °C, in four treatments: 20% egg yolk (T1); 6% (T2); 8% (T3); and 10% LDL (T4). Sperm motility and membrane integrity after 24, 48, 72 and 96 h and the 50% conservation rate of motile spermatozoa (50 M) were evaluated. The 50 M was less for T1 than for the other treatments (P < 0.01), but T2–T4 did not differ (P > 0.05). In Experiment 2, glycerol at 10% was included in the freezing extender, in treatments similar to those from Experiment 1. Sperm motility and membrane integrity did not differ for T2, T3 and T4 at any period in Experiment 1 and after thawing in Experiment 2 (P > 0.05), but were greater for all LDL treatments than for T1 (P < 0.01), in both experiments. Thus, LDL can replace egg yolk in the composition of the TRIS–glucose extender for cooled or frozen dog semen.     

Adaptation of ubiquitin-PNA based sperm quality assay for semen evaluation by a conventional flow cytometer and a dedicated platform for flow cytometric semen analysis

The purpose of semen quality evaluation is to predict the fertility potential of the sample in an objective, rapid and inexpensive manner. However, utilization of sperm quality biomarkers such as ubiquitin and lectin Arachis hypogaea agglutinin (PNA) for flow cytometric semen evaluation might eliminate the need for visual assessment by microscopy. Herein, we demonstrate a robust ubiquitin and PNA-based semen evaluation conducted on a simple, easy to operate, dedicated sperm flow cytometer, EasyCyte Plus (IMV Technologies, L'Aigle, France). Semen samples were collected periodically from two dairy bulls, which were subjected to temporary scrotal insults to induce variable semen quality. Samples were labeled with fluorescently-conjugated anti-ubiquitin antibodies (bind exclusively to the surface of defective sperm) and lectin PNA (binds to acrosomal surface in prematurely capacitated and acrosome-damaged sperm). Fluorescent properties of the samples were measured with a conventional flow cytometer (Becton Dickinson FACScan; Becton Dickinson Corp., Franklin Lakes, NJ, USA) and by the EasyCyte (IMV Technologies) instrument. Data from the two flow cytometers were positively correlated for the percentage of PNA-positive sperm with a damaged acrosome (r = 0.47; P < 0.001) and the percentage of ubiquitin-positive, defective sperm (r = 0.68; P < 0.001). Relative intensities of ubiquitin-induced fluorescence in cells with high ubiquitin levels were also positively correlated (r = 0.90). The proportion of sperm with abnormal morphology was positively correlated with ubiquitin-induced fluorescence measured by EasyCyte (IMV Technologies) (r = 0.63; P < 0.001). These observations provided a rationale for the adaptation of a dual ubiquitin-PNA sperm quality assay for flow cytometric semen evaluation.     

Addition of superoxide dismutase mimics during cooling process prevents oxidative stress and improves semen quality parameters in frozen/thawed ram spermatozoa

High levels of reactive oxygen species (ROS), which may be related to reduced semen quality, are detected during semen cryopreservation in some species. The objectives of this study were to measure the oxidative stress during ram semen cryopreservation and to evaluate the effect of adding 2 antioxidant mimics of superoxide dismutase (Tempo and Tempol) during the cooling process on sperm motility, viability, acrosomal integrity, capacitation status, ROS levels, and lipid peroxidation in frozen and/or thawed ram spermatozoa. Measuring of ROS levels during the cooling process at 35, 25, 15, and 5 °C and after freezing and/or thawing showed a directly proportional increase (P < 0.05) when temperatures were lowering. Adding antioxidants at 10 °C confered a higher motility and sperm viability after cryopreservation in comparison with adding at 35 °C or at 35 °C/5 °C. After freezing and/or thawing, sperm motility was significantly higher (P < 0.05) in Tempo and Tempol 1 mM than that in control group. Percentage of capacitated spermatozoa was lower (P < 0.05) in Tempo and Tempol 1 mM in comparison with that in control group. In addition, ROS levels and lipid peroxidation in group Tempo 1 mM were lower (P < 0.05) than those in control group. These results demonstrate that ram spermatozoa are exposed to oxidative stress during the cooling process, specifically when maintained at 5 °C and that lipid peroxidation induced by high levels of ROS decreases sperm motility and induces premature sperm capacitation. In contrast, the addition of Tempo or Tempol at 0.5 to 1 mM during the cooling process (10 °C) protects ram spermatozoa from oxidative stress.     

Rhodiola sacra aqueous extract (RSAE) improves biochemical and sperm characteristics in cryopreserved boar semen

Although Rhodiola sacra aqueous extract (RSAE) has been used in many studies as an antioxidant, its effects on semen characteristics and its antioxidant properties during cryopreservation of boar sperm have never been evaluated. Semen was collected from five Duroc boars (2-4-year-old) twice weekly and frozen-thawed in extender with RSEA. Motion characteristics were assessed with a computer-aided semen analysis (CASA) system, whereas other sperm quality end points were assessed by routine methods. The effective concentration of RSEA in extender ranged from 4 to 8 mg/L and the effect of RSEA on sperm quality was better in glycerol-free extender than extender containing glycerol (P < 0.05). In frozen-thawed boar semen, there was a direct correlation (P < 0.05) between RSEA concentration and glutathione (GSH) concentrations, mitochondrial activity, and hypoosmotic swelling test (HOST), and an inverse correlation (r = −0.982, P < 0.05) between RSEA concentration and malondialdehyde (all end points were significantly higher at 6 mg/L than in the control group). In summary: (i) the effective concentration of RSEA in extender ranged from 4 to 8 mg/L; (ii) the effect of RSEA on sperm quality was better in extender without glycerol; and (iii) there was a significant correlation between RSEA concentrations and concentrations of GSH and MAD in frozen-thawed boar semen (antioxidant effects of RSEA were concentration-dependent). Further studies are needed to define the active ingredient in RSEA that protects boar sperm against ROS.