Semen characteristics and biochemical composition of cloacal foam of male Japanese quails (Coturnix coturnix Japonica) fed diet incorporated with selenium

An attempt was made to investigate the effect of dietary selenium (Se) on physical and cloacal gland size, foam production, biochemical composition of foam and semen biochemical characteristics of male Japanese quail (Coturnix coturnix Japonica). Two hundred twenty‐five (225)‐day‐old male Japanese quail were randomly distributed to three dietary treatment groups for a period of 20 weeks. Each treatment comprised of three replicates, each containing 25 chicks. Three experimental diets were supplemented with 0, 0.5 and 1.0 mg Se/kg (T₁, T₂ and T_3, respectively), and diet T₁ was considered as control. Sodium selenite was used as the source of selenium. All the birds were provided with feed and water ad libitum. Cloacal foam characteristics, that is cloacal gland index and foam weight, were significantly higher in T₂ group. However, body weight, frequency of foam discharge and testes weight (left and right) did not differ significantly (p > 0.05). Physical characteristics of semen, that is semen volume and sperm concentration, did not differ (p > 0.05) among the Se‐treated groups. The sperm motility, live–dead count and abnormality improved significantly (p < 0.05) in 0.5 mg/Se‐supplemented group compared to 0 or 1.0 mg/Se‐supplemented groups. Similarly, fertility and hatchability percentages were higher (p < 0.05) in 0.5 mg/Se‐supplemented group than in control or 1.0 mg/Se‐supplemented counterparts. The biochemical characteristics of foam in terms of total protein, acid phosphatase (ACP) and nitric oxide did not differ (p > 0.05), while the concentration of glucose was higher (p < 0.05) in 0.5 mg/Se‐supplemented diet. On the other hand, alkaline phosphatase (ALP), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were lower (p < 0.05) in 0.5 mg/Se‐supplemented group compared to control or 1.0 mg/Se‐supplemented groups. From this study, it was concluded that supplementation of 0.5 mg Se/kg diet was beneficial for foam variables, biochemical composition of foam, semen characteristics and fertility in male Japanese quail.     

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.     

Effect of dietary vitamin E and selenium supplementation on semen quality in Cairn Terriers with normospermia

Among others, selenium (Se) and vitamin E (VitE) have been provided to dogs to improve semen quality. However, scientific evidence documenting an effect in dogs is lacking. The aim of this study was to investigate the effect of supplementation of these antioxidants on various ejaculate parameters in a randomized, double‐blinded trial using Cairn Terrier males exhibiting normal seminal quality parameters. Three dogs each were fed a standardized diet and supplemented with 0.1 mg Se, 100 mg VitE or 0.1 mg Se + 100 mg VitE/dog for 3 months. Ejaculate analyses (volume, progressive motility, vitality, morphology, concentration) were performed before inclusion (D0) and after 1, 2 and 3 months (+1, +2, +3). At the same time, glutathione peroxidase (GSH‐PX) and VitE in seminal plasma (SP) and GSH‐PX in blood samples were determined. Vitamin E levels in SP were below the detection limit (1.0 mg/L) in all samples. GSH‐PX in blood (164.0–2794.4 IU/L) and SP (18.4–4326.0 IU/L) was highly variable. Supplementation only significantly affected the total percentage of sperm head abnormalities (p = .011). Time significantly affected the percentage of morphologically abnormal sperm (p = .025), sperm head abnormalities (p = .007), proximal droplets (p = .001) and GSH‐PX in SP (p = .015). Additionally, a significant interaction between time and group was identified for the percentage of membrane‐intact sperm (p = .048), head abnormalities (p = .018), acrosomal defects (p = .043) and proximal droplets (p = .002). Although some effects could be identified for selected parameters, we failed to identify a clear trend about how a 3 months VitE and/or Se supplementation affects semen parameters in normospermic Cairn Terriers.     

Quality of Fresh,Cooled, and Frozen Semen From Stallions Supplemented with Antioxidants and Fatty Acids

This study assessed the effect of oral supplementation with the primary antioxidants and fatty acids involved in spermatogenesis (L-carnitine, selenium, vitamin E, omega-3, and omega-6) on the seminal quality in fresh, cooled, and frozen semen of stallions (n = 8), using a randomized design. The animals were divided into Group I (n = 4) and Group II (n = 4) for a 30-week experiment. The two groups alternated between nutraceutical supplementation and a placebo over the course of the experiment. Semen collections were performed in two sets: once in the middle of the experiment, before the two groups switched treatments, and once at the end. The volume, appearance, sperm concentration, spermatozoa kinetics, and membrane integrity of fresh semen were evaluated. The spermatozoa kinetics and membrane integrity of cooled (for 24, 36, and 48 hours) and frozen semen were also evaluated. No differences were observed in volume, appearance, and sperm concentration between treatment and control. However, compared with placebo, nutraceutical supplementation increased (P < .05) total motility, trajectory speed, as well as plasma and acrosomal membrane integrity in spermatozoa from fresh semen. In cooled semen, nutraceutical treatment also increased (P < .05) total motility, speed, and membrane integrity of spermatozoa compared with the control. In frozen semen, supplementation increased (P < .05) spermatozoa progressive motility and plasma membrane integrity. Our results suggest a positive, synergistic effect of the antioxidant L-carnitine and selenium on spermatozoa kinetics. Similarly, the increase in plasma and acrosomal membrane integrity could be attributed to higher concentrations of polyunsaturated fatty acids (a key cell-membrane component), combined with the prevention of excess lipid peroxidation by antioxidants. In conclusion, supplementation with nutraceuticals containing fatty acids and antioxidants improved the quality of fresh, cooled, and frozen stallion semen. Therefore, nutraceutical use should increase the success of artificial insemination with cooled and cryopreserved semen.     

Influence of vitamin E and vitamin E-selenium combination on arginase activity,nitric oxide level and some spermatological properties in ram semen

The effects of vitamin E and vitamin E-selenium combination on seminal plasma arginase activity and nitric oxide level and some spermatological properties in rams were investigated in this study. For control group, animals were injected intramuscularly with physiological saline. For vitamin E group, rams were injected intramuscularly with 300 mg/ram vitamin E. For vitamin E + selenium group, animals were injected intramuscularly with 5 ml/ram vitamin E + selenium. The semen was collected by artificial vagina at 1st, 4th, 24th, 48th and 72nd hr after administration in each group. Significant decreases in seminal plasma arginase activity (at 1st, 24th and 48th hr), nitric oxide level (at 72nd hr) and abnormal sperm rate (at 1st, 24th and 72nd hr), and significant increases in semen volume (at 24th hr), semen mass activity (at 24th and 48th hr), sperm motility (at 24th, 48th and 72nd hr) and concentration (at 1st hr) were observed in vitamin E group compared with control group. Similarly, significant increase in semen volume (at 1st, 24th and 48th hr), mass activity, (at 48th hr), motility (at 48th and 72nd hr) and concentration (at 4th, 24th and 48th hr), and significant decrements in abnormal sperm rate (at 1st, 24th, 48th and 72nd hr), seminal plasma nitric oxide level (at 1st, 4th, 24th and 48th hr) and semen pH (at 24th and 48th hr) were detected in vitamin E + selenium group in comparison to the control group. As a result, it is suggested that vitamin E and/or vitamin E + selenium applications may improve reproductive performance.     

Impact of Ultra‐Rapid Freezing on the Motility,Morphology, Viability and Acrosome Integrity of Epididymal Cat Sperm Diluted in Sucrose‐Based Extenders

The objective of the present study was to investigate the influence of different sucrose‐based extenders on the motility, morphology, viability and acrosomal integrity of epididymal cat spermatozoa cryopreserved by ultra‐rapid freezing method. Nine cats were castrated, and collected semen was diluted 1 : 1 with Dulbecco`s phosphate‐buffered saline‐BSA1%‐based extender supplemented with different sucrose concentrations (0, 0.25, 0.4 and 0.6 m ). After ultra‐rapid freezing, samples were thawed and sperm motility, morphology, viability and acrosome status were assessed. At thawing, the number of progressively motile (p < 0.01) and morphologically normal (p < 0.01) sperm was higher in the sucrose‐supplemented groups than in the sucrose‐free group. Viability of spermatozoa cryopreserved without sucrose was significantly reduced. In extender supplemented with 0.4 m sucrose, spermatozoa viability showed higher values (57.0 ± 4.7; p < 0.01). No significant differences were detected among groups for sperm acrosome integrity. Results support that cat sperm survive after ultra‐rapid freezing using sucrose as a cryoprotectant, and the best results were achieved when 0.4 m of sucrose was used. This is the first report on sperm ultra‐rapid freezing of cat sperm and further studies on extenders, sperm management or cryovials should be carried out to improve sperm cryosurvival.     

Effect of Albumin and Polyvinyl Alcohol on the Vitality, Motility and Acrosomal Integrity of Canine Spermatozoa Incubated in vitro

Sperm culture media used for in vitro fertilization (IVF) procedures are important factors concerning the viability, motility and acrosomal integrity of spermatozoa. The aim of this study was to investigate the effects of three different sperm diluting media, tissue culture medium (TCM‐199), sperm culture medium (Sp‐TALP) and human tubular fluid (HTF) supplemented with varying concentrations of bovine serum albumin (1, 4 and 6%) or polyvinyl alcohol (0.8%) on the acrosomal integrity, motility and viability of canine spermatozoa. Ejaculates collected from four dogs were diluted in all media and spermatozoa were separated from seminal plasma by the swim‐up technique. Sperm progressive motility was assessed using a phase contrast microscope. Viability and acrosomal integrity were evaluated using a dual stain technique (Giemsa–Trypan blue). The results demonstrated that the number of live canine spermatozoa was similar in culture media supplemented or not supplemented with macromolecules. A minimal concentration of albumin (1%) in the three media showed similar effects on vitality, motility and acrosomal integrity, as had higher concentrations (4 and 6%). The percentage of acrosome‐intact spermatozoa was markedly higher after HTF (94.1%) than after TCM‐199 (70.1%) or Sp‐TALP (71.0%) without supplementation. It is concluded that serum bovine albumin, irrespective of the concentration, preserved sperm viability and function, and HTF is the most suitable medium for preserving the acrosome in canine spermatozoa prepared for in vitro manipulation through short incubation.     

Effects of Dietary Thyme (Thymus vulgaris) and Fish Oil on Semen Quality of Miniature Caspian Horse

Thyme (Thymus vulgaris) is a subshrub from the lamiaceae family with plants that are rich in essential oils and antioxidative phenolic substances. The aim of the study was to investigate the effect of dietary thyme and fish oil supplementation on the semen characteristics of miniature Caspian horse. Sixteen stallions were randomly allocated into four groups and received four different diets: unsupplemented control diet, supplemented with fish oil at 2.5% dry matter intake (DMI), supplemented with fish oil (2.5% DMI), and thyme (0.02% DMI), and supplemented with thyme (0.02% DMI). All experimental diets were formulated according to National Research Council (1998). Semen was collected at 0, 30, 60, and 90 days. The semen samples were cooled and preserved at 5°C. Cooled diluted semen samples were evaluated in vitro by microscopic assessments of chilled sperm motility, acrosomal and other abnormalities (head, midpieces, and tail), viability (evaluated by Eosin–nigrosin), and plasma membrane integrity (evaluated by hypo osmolarity swelling test), and the level of malondialdehyde (MDA) was determined during cool storage 0, 24, and 48 hours after collection. The results showed that total and progressive sperm motility and plasma membrane integrity and functionality in all groups were significantly decreased with increasing storage time. On the other hand, the level of MDA in all groups was significantly increased with increasing storage time. Also, the results showed that most sperm quality parameters in this study were significantly higher in fish oil–thyme and fish oil group compared with thyme and control groups after 24 and 48 hours of storage at 5°C. We concluded that dietary supplementation of fish oil and thyme can improve sperm quality in miniature Caspian stallions during storage in cool condition via increasing total and progressive motility and plasma membrane integrity and functionality. More advances in vitro evaluations and artificial insemination are required to reveal the exact effects of thyme on miniature Caspian stallion sperm quality and its fertilizing ability.     

Effect of selenium and vitamin E addition to the extender on liquid stored capercaillie (Tetrao urogallus) semen quality

Captive breeding has become an important tool in species conservations programmes, maintaining genetic diversity and restoring wild, endangered populations. In order to improve the reproductive efficiency of captive kept capercaillie, the purpose of the study was to determine the effect of selenium and vitamin E addition to semen extender on sperm characteristic during short‐term storage. Ejaculates collected individually from four capercaillie were divided into two parts, diluted threefold with basic EK extender and EK enriched with 1 mg/ml of organic selenium and 8 mg/ml of vitamin E (EK+Se+E) and stored 24 hr at temp. +4°C. Spermatozoa morphology, motility and motility parameter were evaluated in net, diluted and stored semen samples. Significant (p < .05) differences between individual males were stated in relation to the majority of traits evaluated in the freshly collected semen. Comparing to the fresh semen, a significant (p < .05) decrease in percentage of live sperm in total (by 3.8% points on average) has been observed in samples diluted by EK extender, while in semen diluted with EK+Se+E extender this decrease was lower (1.5%pts on average) and not significant. Also per cent of motile sperm in EK+Se+E extender was higher (p < .05) then in EK (71.6% vs. 58.9%), but taking into account the values of individual males, both extender and male effect on liquid semen storage become apparent. Obtained data allow concluding that selenium and vitamin E addition to EK extender had positive effect on morphology and motility of capercaillie semen stored 24 hr at 4°C and can be recommended for similar studies carried out on other Galliformes species.     

Antioxidant supplementation,effect on post‐thaw spermatozoan function in three sturgeon species

High levels of reactive oxygen species (ROS), which may be associated with reduced sperm quality, can be detected during cryopreservation of sperm of some species. Our objective was to investigate whether the addition of antioxidants to cryopreservation extenders influenced post‐thaw sperm characteristics and fertility in Acipenser dabryanus, Acipenser sinensis and Acipenser baerii. Prior to freezing, sperm samples were diluted with a base extender as control or in extender supplemented with catalase (CAT), glutathione (GSH), cysteine (NAC), ascorbic acid (VC) or their paired combinations. Protective concentrations of CAT, GSH and VC in the three species were 25 U/ml, 0.25‐0.5 mg/ml and 0.5 mg/ml, respectively. Cysteine showed no protective effect against ROS. The addition of CAT, GSH and VC positively affected either acrosome or membrane integrity of post‐thawed sperm in the three species, as well as spermatozoan motility in A. sinensis. The combination of antioxidants did not show a positive synergistic effect. This study suggested that the use of antioxidants in the cryopreservation of sturgeon sperm has potential to decrease intracellular ROS, and consequently preserve acrosome and membrane integrity, as well as spermatozoan motility.     

Organic zinc and copper supplementation on antioxidant protective mechanism and their correlation with sperm functional characteristics in goats

Trace minerals feeding had significant effects on sperm production and fertility with better absorption and proper utilization within the body for optimum reproductive function. Several studies have shown that more influenced trace elements in the diets of animals are copper (Cu) and zinc (Zn). Bucks showing deficiency of this mineral might affect the quality of semen production which in turn would affect the fertility. This experiment was thus designed to test the effects of organic Cu and Zn supplementation on antioxidants enzyme activities and sperm functional attributes in fresh semen of bucks. Forty bucks (n = 40, Aged 5 months) were assigned to ten groups of four animals in each group, supplemented (for a period of 8 months) with different levels of organic Zn: 20 mg (T2), 40 mg (T3) and 60 mg (T4), organic Cu: 12.5 mg (T5), 25 mg (T6), 37.5 mg (T7) and combined organic Zn and Cu: 20 + 12.5 mg (T8), 40 + 25 mg (T9), 60 + 37.5 mg (T10), respectively, per kg dry matter and no additional mineral diet (control; T1). One hundred and sixty semen samples were collected through electro‐ejaculator and analysed for sperm quantity, quality, acrosome intactness and plasma membrane integrity and correlated with the catalase, superoxide dismutase, glutathione peroxidase and glutathione reductase enzyme activities in seminal plasma. The results indicated organic Cu and zinc supplemented bucks produced more sperm cells, had higher sperm concentrations, maintained higher (p < .01) sperm livability, plasma membrane and acrosome integrities, more motility and velocity. The increased antioxidant enzyme activities, reduced oxidative stress and lowered lipid peroxidation were positively correlated (p < .05) with the sperm functional attributes. In conclusion, organic Cu and Zn supplement to male goats showed protective roles against oxidative damage and maintained better fresh semen characteristics.     

The Effects of Centrifuged Egg Yolk Used with INRA Plus Soybean Lecithin Extender on Semen Quality to Freeze Miniature Caspian Horse Semen

The aim of this study was to determine the synergistic effects of centrifuged egg yolk (EY) and soybean lecithin on post-thaw Caspian horse sperm motility, morphological abnormalities, and assessment of membrane integrity. The centrifuged EY (CEY) was added at concentrations of 2% and 4% to a defined INRA plus 1.25% soybean lecithin extender used to freeze Caspian horse semen. In this experiment, ejaculates collected from each Caspian horse (n = 4) were divided into three equal aliquots and diluted in CEY 2% (INRA2), 4% (INRA4) supplemented, and without any CEY (INRA0) in INRA plus 1.25% soybean lecithin extender, respectively. Thereafter, samples were frozen and thawed following a standard protocol. Sperm cryosurvival was evaluated in vitro by microscopy assessments of post-thaw sperm motility (by means of computer-assisted semen motility analysis [CASA]), acrosomal and other abnormalities (head, mid-pieces, and tail) and plasma membrane integrity (evaluated by HOST). In Caspian stallion, semen extended with INRA2 had significantly higher CASA motility and CASA progressive motility than those extended with the rest of extenders after freezing and thawing (P < .001). There was no significant difference in path velocity (VAP), VCL, and ALH among three groups (P > .05). For straight line velocity (P < .01) and LIN (P < .001), the highest values were obtained from the INRA4 group. The highest percentages of acrosomal and other abnormalities were found in semen diluted in INRA4 (P < .001). In the group frozen INRA2, the percentage of membrane integrity was significantly higher than that of the other groups (P < .001). The use of CEY 2% in combination with soybean lecithin significantly improved Caspian horse semen freezability.     

Effect of Different Levels of Glycerol and Cholesterol‐Loaded Cyclodextrin on Cryosurvival of Ram Spermatozoa

This study was conducted to determine the optimum level of glycerol and cholesterol‐loaded cyclodextrin (CLC) in a Tris‐based diluent for cryopreservation of ram spermatozoa. Ram semen was treated with 0, 1.5, 3 or 4.5 mg CLC/120 × 10⁶ cells in Tris‐based diluents containing 3, 5 or 7% glycerol in a factorial arrangement 3 × 4 and frozen in liquid nitrogen vapour. Sperm motility, viability (eosin–nigrosin staining) and functional membrane integrity (hypo‐osmotic swelling test) were assessed immediately after thawing (0 h) and subsequently after 3 and 6 h at 37°C. There was an interaction between CLC and glycerol on the functional membrane integrity (p < 0.05). In the presence of 3% glycerol, the highest functional membrane integrity (32.2%) was found in the spermatozoa treated with 1.5 mg CLC/120 × 10⁶ sperm. Post‐thaw sperm motility was highest in 1.5 mg CLC immediately after thawing (40.5%) and after 3‐h (30.6%) incubation at 37°C (p < 0.05). Viability of spermatozoa was higher in all CLC treatments than in the untreated samples, and it was highest (33.9%) in the spermatozoa treated with 1.5 mg CLC (p < 0.05). These data indicate that the addition of cholesterol to sperm membranes by 1.5 mg CLC/120 × 10⁶ cells may allow the use of a lower concentration of glycerol (3%), which is sufficient to mitigate the detrimental effects of freezing and thawing.     

Effects of ascorbic acid 2‐glucoside and alpha‐tocopherol on the characteristics of equine spermatozoa stored at 5°C

The aim of this experiment was to evaluate the effects of adding ascorbic acid 2‐glucoside (AA2G), a water‐soluble antioxidant and stable derivative of ascorbate, to the semen extender and compare it to the addition of vitamin C (Vit. C) and the fat‐soluble antioxidant α‐tocopherol (α‐Toh), both individually and in combination, on the seminal variables of equine sperm submitted to cooling for 72 h. We used two ejaculates from 10 stallions and evaluated them for motility, membrane integrity, chromatin fragmentation, mitochondrial activity and lipid peroxidation. In the analysis of lipid peroxidation, the control group showed 2506.2 ± 796.4 ng malondialdehyde/10⁸ sperm, which was higher (P < 0.05) than the groups treated with antioxidants. The average value of motility in the AA2G group was 68.4 ± 18.1%, which was higher (P < 0.05) than that observed in the control group (62.1 ± 16.2%). The variables membrane integrity, chromatin fragmentation and mitochondrial activity did not show significant difference (P > 0.05) between treatments. It was concluded that the antioxidants protected sperm cells from lipid peroxidation and that AA2G was effective during the cooling process of equine semen at 5°C for72 h, providing increased levels of total motility.     

Trypan Blue/Giemsa Staining to Assess Sperm Membrane Integrity in Salernitano Stallions and its Relationship to Pregnancy Rates

Aim of this study was to test the reliability of Trypan blue/Giemsa staining to evaluate sperm membrane integrity, acrosomal intactness and morphology in stallion to verify whether it could be applied in vitro as useful tool for sperm fertilizing ability. Fertility data on inseminated mares were collected to evaluate the relationship of sperm quality to pregnancy rates. Forty‐one ejaculates were collected from 3 stallions of Salernitano Horse Breed and evaluated for gross appearance, volume, visual motility and membrane integrity with Trypan blue/Giemsa staining and thirty‐five mares were inseminated during the breeding season from April to July. Differences among stallions were found in volume, sperm concentration (p < 0.05) and visual motility (p < 0.01). A decrease in sperm motility, concentration (p < 0.05) and total sperm number was found in June–July (p < 0.01). Live sperm with intact acrosome (LSIA) and proximal droplets (PD) were lower (p < 0.01) in June–July, while acrosome reacted sperm (ARS) percentage increased (p < 0.05). No fertility differences were found among stallions with an average fertility per cycle of 44.6% and a pregnancy rate of 68.6%. Higher percentages of LSIA were found in the ejaculates used to inseminate mares that became pregnant vs those used in mares not pregnant (p < 0.05). The significance of LSIA as test variable to verify the reliability of Trypan blue/Giemsa staining was confirmed by Receiver operating characteristic ROC analysis and the sensitivity of the test was 85% at a cut‐off value of 48% LSIA. Trypan blue‐Giemsa showed to be an accurate method that can be applied on field to evaluate sperm membrane integrity and to identify poor‐quality ejaculates.     

Influence of seminal plasma during different stages of bovine sperm cryopreservation

This study aimed to evaluate the effect of seminal plasma on bovine sperm cryopreservation and to assess the integrity of plasma and acrosomal membranes, mitochondrial potential, remodelling of F-actin cytoskeleton and sperm chromatin fragmentation during the cooling, equilibrium and freezing/thawing stages. Six ejaculates collected from seven Nelore bulls (n = 42) were used in this study. Each ejaculate was divided into two aliquots (with seminal plasma = SP group; without seminal plasma = NSP group) and packed to a final concentration of 50 × 10⁶ sperm per straw. Statistical analyses were performed using SAS software (version 9.3), and p ≤ .05 was considered significant. A time effect was observed for all sperm characteristics (p < .05), except for chromatin fragmentation (p > .05). The presence of seminal plasma better preserved the acrosomal integrity (SP = 75.2% and NSP = 71.7%; p < .05) and also provided lower F-actin remodelling during cryopreservation process (SP = 29.9% and NSP = 32.4%; p < .05). Regarding to the cryopreservation stages, it was observed that cooling step induced higher remodelling of F-actin than the equilibrium and freezing/thawing stages (56.3%, 32.2% and 23.9%, respectively; p < .05). The equilibrium step had minor influence on overall sperm characteristics while the freezing/thawing stage was responsible for the highest percentage of damage in plasma membrane (−65.2%), acrosomal membrane (−34.0%) and mitochondrial potential (−48.1%). On the other hand, none of the cryopreservation stages affected chromatin integrity. It was concluded that the presence of seminal plasma provides increased acrosomal integrity and reduced remodelling of F-actin cytoskeleton. Higher F-actin remodelling is observed after the cooling step while the freezing/thawing step is most damaging to sperm membranes and mitochondrial potential during bovine sperm cryopreservation.     

Effects of Bovine Serum Albumin on Boar Sperm Quality During Liquid Storage at 17°C

This study aimed to investigate the effects of bovine serum albumin (BSA) on boar sperm quality during liquid storage at 17°C. Boar semen samples were collected and diluted with Modena containing different concentrations (0, 1, 2, 3, 4, 5 and 6 g/l) of BSA, and sperm motility, plasma membrane integrity, acrosome integrity, total antioxidative capacity (T‐AOC) activity and malondialdehyde (MDA) content were measured and analysed. The results showed that Modena supplemented with 3, 4 and 5 g/l BSA could improve boar sperm motility, effective survival time and plasma membrane integrity (p < 0.05), decrease MDA content (p < 0.05), while no statistical difference was observed for sperm acrosome integrity and T‐AOC activity among these three groups (p > 0.05). The semen sample diluted with Modena containing 4 g/l BSA could achieve optimum effect, and sperm survival time was 7.5 days. After 7 days preservation, sperm motility, plasma membrane integrity and acrosome integrity were 54%, 49% and 78%, respectively. T‐AOC activity and MDA content were 1.03 U/ml and 17.5 nmol/ml, respectively. In conclusion, Modena supplemented with BSA reduced the oxidative stress and improved the sperm quality of boar semen during liquid storage at 17°C, and 4 g/l BSA was the optimum concentration. Further studies are required to obtain more concrete results on the determination of antioxidant capacities of BSA in liquid preserved boar semen.     

Modulation of seminal plasma content in extended semen improves the quality attributes of ram spermatozoa following liquid preservation at 3–5°C

Seminal plasma (SP) is known to induce motility and capacitation in spermatozoa curtailing their lifespan when preserved. Hence, this study was conducted to examine the effects of removal of SP from sperm surface prior to liquid preservation either by high dilution (1/15) or by washing and the poststorage treatment with SP (15% and 25%, v/v) on the quality attributes of liquid‐preserved ram semen. Over the period of storage, the rapid motility (66.0% and 71.1% vs. 58.3%), straightness (87.1% and 82.1% vs. 79.4%), average path velocity (152.3 and 152.0 µm/s vs. 133.3 µm/s) and the straight‐line velocity (131.3 and 127.8 µm/s vs. 108.5 µm/s) were significantly (p < 0.05) higher in both the high‐dilution and wash groups as compared to the control (1/3 dilution). The functional membrane integrity (82.3% vs. 77.2%) and noncapacitated sperm count (65.0% vs. 58.7%) were also significantly (p < 0.05) higher in the high‐dilution and wash groups, respectively, as compared to the control. The poststorage treatment of sperm with SP significantly (p < 0.05) increased the functional membrane integrity (70.1% vs. 53.8%) and most of the motility attributes as compared to the control (without SP). In conclusion, both the removal of SP prior to liquid preservation and poststorage treatment with SP significantly improved the quality attributes of ram spermatozoa.     

Comparison of two methods of seminal plasma removal on buffalo (Bubalus bubalis) sperm cryopreservation

Cryopreservation causes damage to spermatozoa, and methods minimizing this damage are therefore needed. Although much discussed, seminal plasma removal has become an alternative to improve sperm quality and viability after freezing and has been applied to different species in attempt to obtain good results. The objective of this study was to evaluate semen quality in buffaloes submitted to two methods for seminal plasma removal (filtration and centrifugation). Semen samples were collected from seven Murrah buffalo bulls (Bubalus bubalis) once a week for 8 weeks. Each ejaculate was divided into three groups: control (presence of seminal plasma), centrifugation and filtration. Sperm kinetics was evaluated with the computer‐assisted sperm analysis (CASA) system. Plasmalemma and acrosomal membrane integrity, mitochondrial membrane potential and reactive oxygen species (ROS) were measured by flow cytometry, and lipid peroxidation was evaluated by the thiobarbituric acid reactive substances (TBARS) assay. Seminal plasma removal did not improve sperm kinetics compared to the control group. Centrifugation increased the number of cells with damaged acrosomal membranes (0.77 ± 0.05) and filtration caused greater plasmalemma and acrosomal membrane damage (22.18 ± 1.07). No difference in the mitochondrial membrane potential was observed between groups. In contrast, ROS production was higher in the centrifugation group compared to the control and filtration groups, although no differences in TBARS formation were detected. In conclusion, seminal plasma removal did not improve the quality of thawed buffalo semen compared to control in terms of sperm kinetics, membrane integrity, mitochondrial membrane potential or lipid peroxidation.     

Changes in fertilization medium viscosity using hyaluronic acid impact bull sperm motility and acrosome status

The female reproductive tract, in particular the composition of the uterine and oviduct fluids, is responsible, at least in part, for triggering sperm cell modifications, essential for the acquisition of fertilization ability. Hyaluronic acid (HA) is a glycosaminoglycan present in these fluids, and its role in the fertilization process and sperm functionality is still barely understood. This work was designed to (a) determine the rheological characteristics of the fertilization medium by the addition of HA and (b) determine the HA influence on sperm motility and functional status. To that end, the in vitro fertilization medium was supplemented with 4 doses of HA (6, 60, 600 and 6,000 µg/ml) and analysed for viscosity and adhesion strength characteristics. Then, thawed semen from 6 bulls were incubated in these media and assessed at 4 different moments for morphological and functional parameters (plasma and acrosomal membrane integrities, mitochondrial membrane potential, capacitation, acrosomal reaction, and motility). The rheological evaluation showed that the addition of HA was able to increase both the viscosity and the adhesion strength of the fertilization medium, especially in the 6,000 µg/ml group in which the effect was more pronounced. No influence of HA could be observed on mitochondrial potential, and acrosomal and plasma membrane integrities. However, HA supplementation, at lower doses, led to an increase in the number of reacted sperm, as well as changes in motility parameters, with increase in the number of motile, rapid and progressive spermatozoa. In conclusion, the addition of HA alters the rheological properties of the fertilization medium and leads to the improvement of the properties related to sperm motility and capacitation, without compromising other functional aspects of the cell.