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.     

Antioxidant effect of rosemary (Rosmarinus officinalis) on boar epididymal spermatozoa during cryopreservation

The objective of the present study was to evaluate the ability of rosemary to protect epididymal boar spermatozoa from freeze-thaw damage. Testis from eight boars were collected at the slaughterhouse in two trials. In the laboratory, sperm from epididymis were recovered by flushing and cryopreserved in lactose-egg yolk solution supplemented with various concentrations (low; medium; high) of rosemary. After thawing, total motility, viability, acrosome integrity, response to hypoosmotic swelling test (HOST) and malonaldehide (MDA) concentration were assessed. The results showed that there was an increase in motility at 1, 2 and 3 h in the presence of rosemary. The addition of this herb provided a significant beneficial effect on viability at 2 h of incubation, compared to the control group. Conversely, acrosome status was not affected by any extender. Higher concentration of rosemary produced significant improvement in percentages of positive HOST at 0 and 1 h, whereas no impact was observed at the end of incubation. Considering membrane lipid peroxidation, a greater decrease in MDA production was observed when rosemary content was raised. Rosemary-enriched freezing extender improved the post-thaw epididymis boar spermatozoa quality, showing a significant correlation between rosemary concentration and concentration of MDA. Further studies are needed to define the active component in rosemary that prevents peroxidation.     

Effect of bioactive peptide on ram semen cryopreservation

This present study investigated the effect of bioactive peptide (BAPT) (BAPT) on the quality of ram semen during cryopreservation. Ram ejaculates were extended with Tris buffer supplemented with no antioxidants (as control group), 20 μg/mL BAPT (as BAPT20 group), 40 μg/mL BAPT (as BAPT40 group) and 60 μg/mL BAPT (as BAPT60 group). After cryopreservation, sperm quality including motility, vitality, the percentage of hypoosmotic swelling test (HOST)-positive spermatozoa and the percentage of intact acrosomes was assessed. Furthermore, the malondialdehyde (MDA) in seminal plasma and spermatozoa were analyzed, followed by the measurement of superoxide dismutase (SOD), catalase (CAT) and glutathione-peroxidase (GSH-Px) levels in seminal plasma. After in vitro fertilization, the embryonic cleavage rates and development rates of different groups were analyzed to compare the developmental abilities of spermatozoa. The results showed that the post-thaw sperm motility was significantly higher in the BAPT60 group compared to those in the BAPT20, BAPT40 and control groups (P < 0.05). The percentage of live sperms significantly increased from 48.12 ± 2.35% for the BAPT20 group, 55.43 ± 2.16% for the BAPT40 group to 57.53 ± 3.15% for the BAPT60 group. The percentage of HOST-positive spermatozoa was significantly higher in the BAPT60 group than those in BAPT20, BAPT40 and control groups (P < 0.05). The MDA levels in seminal plasma and spermatozoa were significantly reduced with BAPT supplement (P < 0.05). Additionally, the SOD, CAT and GSH-Px levels in the BAPT experimental groups were significantly higher than those of the control group, which further indicated that BAPT significantly inhibit the reactive oxygen species (ROS) production during the cryopreservation of ram semen. Furthermore, the embryonic cleavage rates and development rates of the BAPT40 and BAPT60 groups were significantly increased in comparison with the BAPT20 and control groups (P < 0.05).In conclusion, BAPT improved the ram sperm quality via inhibiting the ROS production during cryopreservation, and could be applied as a promising supplement for ram semen cryopreservation.     

Cryopreservation of ram semen with antioxidant supplemented soybean lecithin-based extenders and impacts on incubation resilience

The scope of this study was investigation the affects of various antioxidants on 1% soybean lecithin-based semen extenders for ram semen cryopreservation. Ejaculates, collected via electrically stimulated ejaculation, that have a thick consistency, rapid wave motion (3–5 on a 0–5 scale) and >75% initial motility were pooled. The pooled samples were split into four equal aliquots as 5 mM Methionine, 5 mM Cysteamine, 1 mM Cysteine and a sample of antioxidant-free control group. Each sample group was diluted to a ratio of 1/5 (semen/extender, v/v) as final concentration and two step dilution method was used for cryopreservation. Extender groups were assessed for sperm motility, plasma membrane functional integrity using hypoosmotic swelling test (HOST), damaged acrosome using FITC-Pisum sativum agglutinin (PSA-FITC) and DNA integrity using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). Semen samples also incubated for 6 h in humidified air with 5% CO₂ at 39 °C to evaluate post-thaw incubation resilience of semen characteristics. The results showed that freezing and thawing procedures had negative effects on motility (P < 0.05), plasma membrane integrity (P < 0.05) and acrosomal integrity (P < 0.05). After 6 h of incubation time, the Cysteine supplemented extender group yielded significantly higher results than other extender groups in terms of spermatological parameters. Furthermore MDA levels in the antioxidant groups were lower than control group (P < 0.05). Nevertheless, there were no significant differences among antioxidant groups.     

Effects of reduced glutathione and catalase on the kinematics and membrane functionality of sperm during liquid storage of ram semen

The objective of this study was to evaluate the effects of reduced glutathione (GSH) and catalase (CAT) supplementation on the kinematics and membrane functionality of sperm during the liquid storage of ram semen, cooled at 5 °C, for up to 24 h. Semen samples from four rams were pooled, diluted with Tris-egg yolk extender without antioxidants (control) or supplemented with either CAT (100, 200, and 400 U/mL) or GSH (100, 200, and 400 mM) at a final concentration of 50 × 10⁶ sperm/mL. Sperm kinematics, which was analyzed by computer-assisted sperm analysis (CASA), and membrane functionality, which was analyzed using the hypo-osmotic swelling test (HOST), were determined after the addition of the semen samples at different processing times (fresh/diluted, 1.5, 6, 12, and 24 h, at 5 °C). No significant differences were recorded in the kinematics or membrane functionality between treatments at different times. The supplementation of diluents with 100 and 200 U/mL of CAT prevented the harmful effects of cooling on total sperm motility. No significant differences were observed in progressive sperm motility throughout processing, regardless of the treatment and time of evaluation. Supplementation with 400 mM GSH resulted in an earlier reduction (P < 0.05) of total sperm motility, a decrease in rapid sperm rate and a reduction in curvilinear velocity during incubation, at 5 °C. The cooling induced a reduction (P < 0.05) in the percentage of sperm with a functional plasma membrane (HOST), especially after 1.5 h of incubation. Based on the results of the present study, the addition of CAT (100 and 200 U/mL) reduced the deleterious effects of cooling on total motility in ram sperm maintained at 5 °C for 24 h, although it did not affect the functionality of the sperm membranes. However, the addition of 400 mM GSH caused negative effects on the velocity parameters of the sperm.     

The effect of supplementation of cryopreservation diluents with sugars on the post-thawing fertility of ram semen

This study was conducted to elucidate the effect of increasing the osmolality of a basic Tris, extender supplemented with sucrose, trehalose or raffinose on post-thawing ram semen quality (sperm motility, viability, acrosome integrity, total sperm abnormalities and membrane integrity). After primary evaluation of the collected ejaculates, only semen samples with more than 70% motile sperm, and a sperm concentration of higher than 3 × 10⁹ sperm/ml were used for cryopreservation. The semen samples were pooled and diluted (1:4) with a Tris-citric acid-fructose-yolk extender, supplemented with different concentrations (50, 70 or 100 mM) of sucrose, trehalose or raffinose. As control, semen was diluted and frozen in the base diluent, without additional sugars. Pooled semen samples were aspirated into 0.25 ml straws, cooled to 5 °C within 90 min and frozen by exposure to liquid nitrogen vapor (4-5 cm above the liquid nitrogen surface) for 10 min - before plunging into liquid nitrogen, for storage. After 24 h, straws were thawed in a water bath (37 °C) for 30 s. The frozen-thawed sperm characteristics were improved significantly (P < 0.05) by increasing the level of the sugars. Optimal results being obtained with 70 and 100 mM trehalose or raffinose. All extenders containing supplemental sugars were superior in terms of sperm quality to the control (P < 0.01) group. The highest sperm motility (60.6 ± 1.9%), viability (60.6 ± 2.5%) and membrane integrity (58.2 ± 2.1%) were recorded using 100 mM trehalose and the lowest with 50 mM sucrose (48.6 ± 1.9%, 51.4 ± 2.5% and 47.9 ± 2.1%, respectively). All sugar concentrations decreased the percentage of acrosomal and total sperm abnormalities (P < 0.05). The extenders containing 100 mM trehalose or raffinose significantly (P < 0.05) decreased the occurrence of sperm abnormalities, compared to the other treatments. The fertility rates obtained after cervical insemination of the frozen-thawed sperm were 46.8%, 44.1% and 16.7% for 100 mM trehalose, 100 mM raffinose and the control with supplementation of the diluents, respectively. The study showed that ram sperm can tolerate hyperosmotic diluents, and that a range of sugar concentrations (50-100 mM) may successfully be incorporated in the ram semen cryopreservation diluents, although further research is warranted.     

Influence of the thawing rate on the cryopreservation of semen from collared peccaries (Tayassu tajacu) using Tris-based extenders

The objective was to evaluate the influence of the thawing rate on the quality of frozen-thawed (cryopreserved in Tris-based extenders) semen obtained from collared peccaries (Tayassu tajacu). Semen from 13 sexually mature collared peccaries males were collected by electroejaculation, and evaluated for motility, vigor, sperm viability, membrane integrity, and sperm morphology. Semen was divided in two equal portions: the first was diluted in Tris-fructose and the other in Tris-glucose, with egg yolk (20%) and glycerol (3%) added to each portion. Extended semen was frozen in liquid nitrogen and thawed using two thawing protocols (37 °C for 1 min or 55 °C for 7 s, followed by an additional 30 s at 37 °C). There were no significant differences between the two extenders after extension, chilling, or glycerol addition. After thawing at 37 °C, there were 37.9 ± 4.2% and 28.5 ± 5.1% motile spermatozoa for samples extended in Tris-fructose and Tris-glucose, respectively, with 33.8 ± 3.7% and 28.2 ± 3.5% motile spermatozoa after thawing at 55 °C (no significant differences). Furthermore, there were no significant interactions between extenders and thawing protocols for any semen end point. In conclusion, semen from collared peccaries was successfully cryopreserved in Tris-based extenders and thawed with two protocols (37 °C for 1 min or 55 °C for 7 s).     

Surface changes associated with ram sperm cryopreservation revealed by counter-current distribution in an aqueous two-phase system effect of different cryoprotectants

Ram sperm was frozen in the presence of the most commonly used cryoprotectants. After thawing, the overall cell surface changes provoked by freezing were assessed by centrifugal counter-current distribution (CCCD). In addition, cell membrane integrity (viability) of all the treated sperm was estimated by fluorescent staining. Fresh and refrigerated sperm were used as controls. Our results show no improvement of the cooling-induced cell surface damage by freezing in the presence of bovine seminal plasma, proline, glycine-betaine and phosphatidylcholine. Better results were obtained with vitamin E and cholesterol. However, the best protective effects were found by employing seroalbumin and lactalbumin. Furthermore, freezing in the presence of bovine lactalbumin resulted in a good maintenance of the cellular viability and of the CCCD heterogeneity in respect to fresh cells.     

Effect of semen dilution to low-sperm number per dose on motility and functionality of cryopreserved bovine spermatozoa using low-density lipoproteins (LDL) extender: Comparison to Triladyl and Bioxcell

Artificial insemination with doses containing low-sperm numbers has been utilized to optimize the use of elite bulls. Hen egg yolk is widely used as a cryoprotective agent in semen freezing extender protecting the spermatozoa. Its action is due to the presence of low-density lipoproteins (LDL) in the hen egg yolk. The objectives of the present study were to evaluate the effects of the semen dilution to low-sperm number/dose on sperm motility and integrity of sperm plasma membrane in the cryopreservation process, using two commercial extenders (Triladyl^®, Bioxcell^®) and LDL extender prepared in our laboratory, 97% purity. Fifteen ejaculates were collected from five fertile crossbred bulls (Bos taurus × Bos indicus). After collection, sperm motility was examined by Computer-Assisted Semen Analysis (Hamilton Thorne), morphological sperm characteristics were evaluated by differential interference microscopy and the integrity of plasma membranes was determined using the hypo-osmotic swelling test. The semen was subsequently divided into three aliquots and diluted with the three extenders into 120 × 10⁶, 60 × 10⁶ and 20 × 10⁶ sperm/mL, corresponding to 30 × 10⁶, 15 × 10⁶ and 5 × 10⁶ sperm/dose, respectively. This study revealed that LDL extender was more effective in preservation of motility and integrity of the plasma membrane of spermatozoa than Bioxcell^® and Triladyl^® (p < 0.05), but no significant difference was observed between Triladyl^® and Bioxcell^®. Therefore we can conclude that LDL extender could be used instead of Triladyl^® or Bioxcell^® at low semen concentration per dose for elite bulls, it also could be envisaged for the industry of sex-stored semen.     

Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of ram seminal plasma proteins and their correlation with semen characteristics

The present study was conducted to investigate fertility-associated proteins in ram seminal plasma and the correlation between specific protein and semen characteristics in sheep. Thirty-eight German merino sheep clinically proven healthy were chosen and divided into three groups according to fertility. Ejaculates were collected by an artificial vagina and semen characteristics (volume, pH value, motility, viability and concentration) were recorded. Seminal plasma was harvested by centrifugation and then subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis in parallel with molecular weight standards. Fifteen protein bands with different molecular weights, ranging from 15.13 to 116.20 kDa, were identified on the gel. The results showed that the relative content of eight protein bands was significantly different between the high-fertility group (H-group) and the low-fertility group (L-group). Although the remaining seven protein bands showed no fertility-associated changes in their relative content, some of them were negatively or positively correlated with some semen quality parameters (motility, viability, concentration or pH value). Thus, this study indicates that ram seminal plasma contains specific proteins that are associated with fertility and semen characteristics. Also, these proteins could be utilised in developing a reliable and simple method to determine the ram fertility or semen quality.     

Effects of water-soluble Laminaria japonica polysaccharide 3 (LJP-P3) on bull cryopreservation sperm

In this study, water-soluble Laminaria japonica polysaccharide3 (LJP-P3) was investigated for the cryoprotective effects on bull sperm. Five concentrations of LJP-P3 with 0.1, 1, 10, 50 and 100 mmol/L were added into the extenders of bull semen, respectively, and the effects on quality of sperm after freezing-thawing were assessed. The results showed that the kinematic parameters of bull sperm including linear motile sperm (LM), curvilinear line velocity (VCL) value, straight line velocity (VSL) and velocity of the average path (VAP) were greater in the extenders containing LJP-P3 (P<0.05). In comparison to those of other treatments and control group the extenders containing 1.0, 10.0 and 50.0 mmol/L of LJP-P3 led to higher percentage of mitochondrial activity and sperm membrane integrity(P<0.05), and the acrosome integrity of bull cryopreservation sperm were significantly improved in all treatment groups. Moreover, the higher GSH-Px, SOD and CAT levels in bull cryopreservation sperm were favored from the extenders of 10.0, 50.0 and 100.0 mmol/L LJP-P3 added (P<0.05) compared with other treatments and control group. In addition, the results of artificial insemination showed that both the pregnancy rate and the number of calving were higher in the group of semen containing 10 mmol/L of LJP-P3 than that of control group (P < 0.05). In summary, LJP-P3 exhibited a greater cryoprotective effect to bull sperm and the most suitable concentration of LJP-P3 is 10.0 mmol/L.     

Effect of glutamine on post-thaw motility of bull spermatozoa after association with LDL (low density lipoproteins) extender: Preliminary results

Glutamine has been used in the composition of semen extenders in several species, but never in the bull. The aim of our study is to demonstrate the cryoprotective role of glutamine for freezing bovine semen and to determine concentration of the latter to improve the motility and trajectory characteristics of spermatozoa. Three experiments were undertaken with 21 ejaculates from three different bulls. In the first experiment, glutamine was added to 40, 80, and 120 mM of basic medium (BM) which consisted of Tris + glycerol 6.4% (v/v). In the second experiment glutamine was added to the 8% low density lipoprotein (LDL) diluent at 40, 80, and 120 mM. In the third experiment, the best concentration of glutamine was determined; this was then added to the LDL extender at 10, 20, 30, and 40 mM. The semen was diluted then frozen in the different media. Motility parameters were assessed using an image analyser following thawing. Experiment 1 demonstrated that glutamine had a cryoprotective effect; at 40 mM it gave superior motility parameters to those obtained with the basic medium (p < 0.05). Experiment 2 demonstrated that the combination of LDL-glutamine 40 mM and 80 mM did not improve motility and even deteriorated it in comparison with the glutamine-free LDL extender. Experiment 3 demonstrated that the addition of 10 mM of glutamine to the LDL medium lead to a significant improvement (p < 0.05) in the motility of bull spermatozoa and could be used to improve bovine semen extenders.     

Effects of cryopreservation on head morphometry and its relation with chromatin status in brown bear (Ursus arctos) spermatozoa

The Cantabrian brown bear (Ursus arctos) is a highly endangered species in Spain and basic studies are necessary in order to bank its germplasm. Sperm heads are mainly made up of chromatin, thus their shape depends partly on chromatin structure. Thawed semen from 10 bears was used to analyze chromatin status by sperm chromatin structure assay (SCSA) and head morphometry by the computer-assisted sperm morphology assessment (CASMA) system. Morphometry was analyzed before and after freezing–thawing in order to evaluate the effects of cryopreservation on sperm heads. Each spermatozoon was measured for four primary parameters (length, L; width, W; area, A; perimeter, P) and derived parameters (ellipticity: L/W, circularity: 4πA/P², elongation: (L − W)/(L + W), regularity: πLW/4A). All the derived parameters significantly differed between bears. Likewise, cryopreservation affected head morphometry by reducing its size. Clustering based on morphometric parameters separated three subpopulations, one of them being significantly more influenced by the cryopreservation process. We obtained high correlations between head morphometry and SCSA parameters: standard deviation of DNA fragmentation index (SD-DFI) was correlated with perimeter and area (r = 0.75 and r = 0.62, respectively) and DFIm and DFIt (moderate and total DNA fragmentation index) were correlated with perimeter (r = 0.65 and r = 0.67, respectively). Nevertheless, classification of males according to SCSA or head morphometry did not completely agree so the two assays might explain male variability differently. We conclude that cryopreservation affected morphometry at least in a subset of spermatozoa. These results might improve future application of sperm banking techniques in this species.     

Total and specific activities of superoxide dismutase (SOD) in seminal plasma are related with the cryotolerance of jackass spermatozoa

This study investigated whether the activities of four antioxidant enzymes present in jackass seminal plasma (SP), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) and glutathione reductase (GSR), are related to the sperm ability to withstand cryopreservation. Eighteen ejaculates from 16 healthy jackasses were collected and split into two aliquots. The first one was centrifuged (3,000×g, 4 °C for 10 min) and used to determine the activities of these four enzymes in SP, whereas the other was diluted in a skim-milk extender and then cryopreserved. Assessment of sperm motility and membrane integrity was performed before and after cryopreservation. Based on the percentages of total motile and viable spermatozoa at post-thaw, samples were classified as good (GFE) or poor (PFE) freezability ejaculates through cluster analyses. Total and specific activities of SOD in seminal plasma were higher (P < 0.05) in GFE than in PFE, whereas no significant differences between GFE and PFE were observed regarding total and specific activities of CAT, GPX and GSR. However, post-thaw sperm parameters were positively correlated with total and specific activities of CAT and negatively correlated with those of GSR. In conclusion, determination of total and specific activities of SOD in the seminal plasma of a given jackass ejaculate may predict the sperm ability to withstand cryopreservation. In addition, our results warrant further research on addressing whether SOD activity in seminal plasma does not only allow predicting the sperm cryotolerance of a given ejaculate but also that of all ejaculates from a given jackass.     

Effects of glycine and alanine on short-term storage and cryopreservation of striped bass (Morone saxatilis) spermatozoa

Three experiments were designed to examine the effects of the amino acids glycine and alanine on short-term storage and cryopreservation of striped bass spermatozoa. In the first experiment, the effect of glycine on post-equilibration motility was evaluated. In the presence of 2.5 or 5.0% Me(2)SO, glycine treatments (25, 50, and 75 mM) yielded higher (P<0.05) post-equilibration motility at all equilibration times examined compared to the control. There was no difference (P>0.05) among these three glycine treatments. In the second experiment, glycine and alanine at concentrations of 25, 50, 75, or 100mM were evaluated for post-thaw motility in the presence of 2.5 or 5% Me(2)SO. When compared to the control, both the glycine and alanine treatments showed positive effects on post-thaw motility at all concentrations tested. The highest (P<0.05) post-thaw motility was achieved with 50mM glycine or 75 mM alanine using 5% Me(2)SO. No interaction (P>0.05) between Me(2)SO and glycine or alanine was observed, indicating that the effect of glycine or alanine was independent of the concentrations of Me(2)SO. In the third experiment, glycine was evaluated for sperm motility, after short-term refrigerated storage and after cryopreservation of the same refrigerated semen. Sperm motility decreased after 24h of refrigerated storage in 50mM glycine treatment and the control, when compared to fresh sperm motility. However, 50mM glycine treatment yielded higher (P<0.01) sperm motility after both 24 and 48 h of storage as well as higher (P<0.01) post-thaw motility when compared to the control. An average of 30+/-2.9% and 16+/-2.4% post-thaw motility was achieved with the 50mM glycine treatment after 24 and 48 h of refrigerated semen, respectively.     

The effects of clove oil on the enzyme activity of Varroa destructor Anderson and Trueman (Arachnida: Acari: Varroidae)

Varroa destructor, a key biotic threat to the Western honey bee, has played a major role in colony losses over the past few years worldwide. Overuse of traditional acaricides, such as tau-fluvalinate and flumethrin, on V. destructor has only increased its tolerance to them. Therefore, the application of essential oils in place of traditional pesticides is an attractive alternative, as demonstrated by its high efficiency, lack of residue and tolerance resistance. To study the acaricidal activity of essential oils, we used clove oil (Syzygium aromaticum L.), a typical essential oil with a wide range of field applications, and examined its effects on the enzyme activities of Ca²⁺-Mg²⁺-ATPase, glutathione-S-transferase (GST) and superoxide dismutase (SOD) and its effects on the water-soluble protein content of V. destructor body extracts after exposure to 0.1 μl and 1.0 μl of clove oil for 30 min. Our results showed that the water-soluble protein content significantly decreased after the treatments, indicating that the metabolism of the mites was adversely affected. The bioactivity of GSTs increased significantly after a low dosage (0.1 μl) exposure but decreased at a higher dosage (1.0 μl), while the activities of SOD and Ca²⁺-Mg²⁺-ATPase were significantly elevated after treatments. These results suggest that the protective enzyme SOD and detoxifying enzymes Ca²⁺-Mg²⁺-ATPase and GST contributed to the stress reaction of V. destructor to the essential oils and that the detoxification ability of V. destructor via GST was inhibited at higher dosages. Our findings are conducive to understanding the physiological reactions of V. destructor to treatment with essential oils and the underlying mechanisms behind the acaricidal activities of these natural products.     

Effects of cholesterol-loaded cyclodextrin during freezing step of cryopreservation with TCGY extender containing bovine serum albumin on quality of goat spermatozoa

The susceptibility of mammalian spermatozoa to cold shock and freezing damage is due to changes in membrane lipid composition, particularly cholesterol depletion in plasma membrane during cryopreservation. The aim of this study was to investigate the effects of different concentrations of cholesterol-loaded cyclodextrin (CLC) and bovine serum albumin (BSA) on the cryopreservation of goat spermatozoa in tris-citrate egg yolk extender. Semen was collected from four mature goats and divided into seven aliquots prior to cryopreservation. The first aliquot remained untreated and was mixed with TCG, the second aliquot was mixed with TCG and egg yolk (TCGY), third aliquot was mixed with TCGY and 2.5% BSA (TCGYB) and other aliquots were mixed with TCGYB containing 0.75, 1.5, 2.5 and 3 mg/ml CLC. All samples were cryopreserved in straws over liquid nitrogen vapor and sperm motion Kinetics were measured by computer-assisted semen analysis (CASA) (percent motility (MOT), curvilinear velocity (VCL), straight-line velocity (VSL), average path velocity (VAP), linearity (LIN), and amplitude of lateral head displacement (ALH)). Acrosome status and vitality was observed by the triple-stain technique. CLC addition to extender resulted in significant (p < 0.05) enhancement of MOT, STR, and VCL of post-thawing sperm. Post-thawed motility, progressive motility and recovery rate were significantly (p < 0.05) higher in 1.5 mg/ml CLC with 2.5% BSA in TCGY extender compared to other groups. The 1.5 CLC sperm yielded a significant increase in percentage of spermatozoa with intact acrosome (P > 0.05). These results indicate that treating goat sperm with CLC and BSA in TCGY extender improved motility and vitality after freezing and thawing.     

The effects of clove oil on coral: An experimental evaluation using Pocillopora damicornis (Linnaeus)

Clove oil solution (10% clove oil, 90% ethanol) is an anaesthetic that is widely used to catch demersal fish on coral reefs. This study assessed the effects of clove oil solution on colonies of Pocillopora damicornis, a cosmopolitan reef coral. In the laboratory, low concentrations (0.5 ppt) of clove oil solution had no effect on coral colour or photosynthetic efficiency, irrespective of exposure time (1-60 min). Corals treated with high concentrations (50 ppt) of clove oil solution died immediately, including those that were exposed briefly (1 min). Intermediate concentrations (5 ppt) of clove oil solution produced variable results: a 1 min exposure had no effect, a 10 min exposure caused bleaching and reduced photosynthetic efficiency, and a 60 min exposure caused total mortality. To validate these observations, clove oil solution was applied to corals in situ. Sixty-three days after application, corals treated with 10 ml of clove oil solution appeared to be unaffected. It was concluded that (1) limited amounts of clove oil solution are unlikely to harm this coral, and (2) clove oil solution may represent an ‘eco-friendly’ alternative to cyanide for use in the live reef-fish trade.     

Effect of different olive oil-derived antioxidants (hydroxytyrosol and 3,4-dihydroxyphenylglycol) on the quality of frozen-thawed ram sperm

The aim of the present study was to evaluate the effect of the addition of different concentrations of two olive oil-derived antioxidants, hydroxytyrosol (3,4-dihydroxyphenylethanol, HT) and 3,4-dihydroxyphenylglycol (DHPG), on ovine semen during the freezing-thawing process. Sperm was collected, pooled and diluted with commercial extenders and then divided into aliquots supplemented with different concentrations (10 μg/ml, 30 μg/ml, 50 μg/ml and 70 μg/ml) of HT, DHPG and a mixture (MIX) of both antioxidants. A control group, without antioxidant, was also prepared. Sperm motility, viability, acrosome integrity, mitochondrial membrane potential and lipid peroxidation (LPO) were assessed. The results showed that frozen-thawed ram spermatozoa exhibited lower values for motility, membrane integrity, acrosome and mitochondrial membrane potential than fresh samples (P ≤ 0.01). However, when antioxidants were added, thawed spermatozoa exhibited relatively low LPO, recording values similar to fresh spermatozoa; by contrast, the control group of frozen-thawed spermatozoa without antioxidants exhibited significantly higher LPO (P ≤ 0.01). The addition of a HT+DHPG mixture (MIX) had a negative impact on sperm membrane and acrosome integrity, suggesting that a pure antioxidant supplementation has the potential to offer superior results. In conclusion, HT and DHPG exhibited a positive effect on the frozen-thawed spermatozoa inasmuch as they reduced the LPO. These olive oil-derived antioxidants have the potential to improve frozen-thawed sperm quality, although further studies should be carried out to analyse the antioxidant effect at different times after thawing.     

Influence of different extenders on morphological and functional parameters of frozen-thawed spermatozoa of jaguar (Panthera onca)

Due to the global decrease in jaguar population, conservation strategies are essential and the development of effective semen cryopreservation protocols would contribute to the formation of germplasm banks. Therefore, the objectives were to (1) evaluate the use of TRIS and ACP-117c extenders for jaguar semen freezing, (2) describe the ultrastructural changes in sperm after cryopreservation, and (3) evaluate the binding capacity of the thawed sperm. Eight ejaculates from five mature individuals were collected by electroejaculation, extended in TRIS or a coconut based-extender (ACP-117c), and frozen in liquid nitrogen. Samples were evaluated for sperm motility, vigor, membrane functionality, mitochondrial activity, morphology (using light microscopy, scanning electron microscopy - SEM and transmission electron microscopy - TEM), sperm kinetic parameters (by computerized analysis - CASA), and sperm binding capability using an egg yolk perivitelline membrane assay. Samples preserved in TRIS presented better post-thaw motility (46.0 ± 7.7%) and membrane functionality (60.5 ± 4.2%) and higher mitochondrial activity (21.5 ± 3.7%) than those preserved in ACP-117c (20.9 ± 5.4% motile sperm; 47.1 ± 2.5% functional membrane; 11.8 ± 1.7% mitochondrial activity). Regarding ultrastructural evaluations, SEM showed that both extenders were able to preserve the superficial membrane of the sperm, but TEM revealed the occurrence of nuclear electron lucent points, especially in samples extended in ACP-117c. Additionally, TRIS also provided a higher number of sperm bound to the perivitelline membrane (29.5 ± 3.3%) in comparison to samples diluted in ACP-117c (18.6 ± 1.5%). Overall, we suggest the use of a TRIS-based extender for cryopreservation of jaguar semen.