Chilled storage of Asian seabass,Lates calcarifer Bloch semen: Effects of ions,extenders and storage periods on sperm quality and fertilization ability

The development of a chilled storage protocol of fish sperm requires an understanding of sperm biology and function as the activation/inhibition of fish sperm is greatly affected by several environmental factors. This study aimed to determine the effects of ionic and non-ionic solutions, extender types (Ringer's solution, Ca-F HBSS solution, HBSS solution, He and Wood solution, Saline solution, and Modified Cortland solution), and chilled-storage period on sperm quality and fertilization ability of Asian seabass, Lates calcarifer semen. Regulation of Asian seabass sperm motility was dependent on the osmolality of both ionic and non-ionic activation media. The threshold levels on the initiation of sperm motility were detected in KCl (>100 mM), NaCl (>50 mM), CaCl₂ (>50 mM), glucose (>300 mM), and mannitol (>100 mM) solutions. Relatively high percentages of sperm motility (>80%) were observed when activated with KCl, NaCl, CaCl₂, glucose, and mannitol solutions at above 700, 600, 350, 1,000, and 1,000 mM, respectively. Ringer's solution was the most optimal extender for chilled storage of Asian seabass semen at 2–4°C supported by the retention of sperm motility and viability for 6 days. Semen diluted in Ringer's solution and chilled-stored for 2 days exhibited acceptable fertilization (66.1% ± 6.2%) and hatching (56.4% ± 2.9%) rates. This report, for the first time, describes the ionicity and non-ionicity effects on the motility of Asian seabass sperm.     

Basic physico-chemical parameters of milt from sea trout (Salmo trutta m. trutta), brook trout (Salvelinus fontinalis) and rainbow trout (Oncorhynchus mykiss)

The aim of the study was to compare the physico‐chemical parameters of milt from sea trout (Salmo trutta m. trutta), brook trout (Salvelinus fontinalis) and rainbow trout (Oncorhynchus mykiss). Milt was collected by stripping and spermatozoa concentrations, were determined and compared with sperm motility and spermatocrit values along with seminal plasma indices (pH, osmolality, sodium, potassium, chlorine, calcium, magnesium, glucose and protein concentrations). The highest spermatozoa concentration of 22.3 ± 6.7 × 10⁹ ml^?1 was found in the sea trout milt, and was significantly different of those observed in brook trout (11.9 ± 3.3 × 10⁹ ml^?1) and rainbow trout (10.7 ± 4.4 × 10⁹ ml^?1). The values for pH and K⁺ did not differ significantly among species. The mean pH was 8.0 in the milt of each species and the K⁺ concentrations ranged from 24.8 ± 7.2 to 30.5 ± 7.6 mm L^?1. Considerable differences were determined for the Ca²⁺ ions concentrations. The highest value was found in sea trout (1.7 ± 0.3 mm L^?1), while in the rainbow trout it was 0.7 ± 0.5 and in the brook trout 0.4 ± 0.1 mm L^?1. The most pronounced differences were found in the glucose concentration cause of its unnaturally low concentration in rainbow trout of the mean value of 6.0 ± 15.2 mg L^?1. The mean value in sea trout and brook trout was 185.0 ± 172.4 and 231.2 ± 148.4 mg L^?1 respectively. For all species, protein mean values were below 1.3 g L^?1. The mean osmolality was between 230.6 ± 98.6 and 272.0 ± 26.4 mOsm kg^?1 in the species studied. No correlation was found between any components determined in milt and the spermatozoa motility (P > 0.05). The sperm concentration was positively correlated with the protein content in the milt of the three species studied, other less exhibited correlation was found.     

Prostaglandins in rainbow trout (Oncorhynchus mykiss Walbaum, 1792) sperm biology – searching for answers

The purpose of this study was to determine the concentrations of prostaglandins E₂ and F_2α (PGE₂ and PGF_2α) in the blood, testis and seminal plasma of mature male rainbow trout and in the ovarian fluid to assess the effects of these prostaglandins on sperm motility parameters when present in activation media. Also prolonged incubation with prostaglandins on sperm motility and calcium influx were studied. The profile of PGE₂ and PGF_2α differed in concentration between blood, testicular supernatant and seminal plasma. PGE₂ was predominant in the blood sample (0.29 ng ml^?1) and testicular supernatant (3.1 ng ml^?1) whereas their level in seminal plasma was lower than PGF_2α (0.23 ng ml^?1). The concentrations of PGF_2α in blood, testis and seminal plasma were 0.04, 0.99, 1.3 ng ml^?1, respectively. In the ovarian fluid the concentrations of both prostaglandins were higher than in the male reproductive tract. Adding both prostaglandins to activation buffer (at concentrations 15 and 70 ng ml^?1) had no effect on any CASA parameters. Calcium influx related to rainbow trout sperm incubations with PGE₂, and PGF_2α was not detected. After 24 h incubation of sperm in artificial seminal plasma solution without and with prostaglandins all sperm samples increased their motility potential and intracellular calcium concentration. Therefore, this effect was not related to the presence of prostaglandins. In summary PGE₂, and PGF_2α were present in the rainbow trout male reproductive tract, and their profile varies from that of blood, testis and seminal plasma. The specific role of both prostaglandins in salmonid sperm biology remains unclear.     

Ions and osmolality on post‐thaw sperm motility activation of the endangered Brycon insignis (Characiformes)

The aim of this study was to evaluate the effects of osmolality and the presence of ions on the activation of post‐thaw sperm motility of Brycon insignis. Sperm was frozen under a standardized methodology for this species. In experiment 1, 11 solutions were prepared with reverse osmosis (RO) water (～0 mOsm/kg) and glucose or NaCl adjusted to an osmolality of 50, 100, 150, 200 and 250 mOsm/kg. In experiment 2, six solutions were prepared with RO and adjusted to ~100 mOsm/kg with one of the following chemicals: NaHCO₃, sodium citrate (Na₃C₆H₅O₇), NaCl, KCl, CaCl₂ or glucose (as ion‐free control). Post‐thaw sperm of both experiments was evaluated for motility rate, velocities (curvilinear = VCL, among others) and beat‐cross frequency (BCF). In experiment 1, sperm motility rate and velocities were higher (p < 0.05) when triggered in solutions at osmolalities from 0 to 200 mOsm/kg (62–80% motility; 139–167 µm/s) than that at 250 mOsm/kg (36–44% motility; 94–99 µm/s VCL). BCF was not affected by osmolality and varied from 19 to 24 Hz in all samples. In experiment 2, samples activated in NaHCO₃, citrate, NaCl and KCl solutions yielded higher motility rates (76–85%) and BCF (24–25 Hz) compared to those activated in CaCl₂ (50%; 14 Hz). Samples activated in ion‐free control solution yielded higher motility rate (87%) than those activated in NaHCO₃ and in CaCl₂. Curvilinear velocity was higher in samples activated in NaHCO₃, citrate, KCl and control solutions (144–160 µm/s) than in those activated in CaCl₂ (104 µm/s); samples activated in NaCl yielded intermediate VCL values (127 µm/s). Post‐thaw sperm achieves maximum motility rate and velocities when activated in solutions composed of sodium citrate, NaCl, KCl or glucose. Thus, post‐thaw sperm motility of B. insignis can be triggered in ionic and non‐ionic solutions at osmolality between 0 and 200 mOsm/kg. The use of solutions containing calcium, however, should be avoided.     

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

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

Optimization of short-term storage of pikeperch semen: an applicable approach

Contamination of semen with urine and asynchronous maturation of males and females are main obstacles in artificial reproduction of pikeperch Sander lucioperca. The objective of this study was to overcome these obstacles using optimization of a procedure for short-term storage of pikeperch semen at 4 °C using two immobilizing media (IM): (a) IM1, 180 mM NaCl, 2.68 mM KCl, 1.36 mM CaCl₂ ⋅ 2H₂O and 2.38 mM NaHCO₃, 343 mOsm/kg; and (b) IM2, 200 mM NaCl, 2.68 mM KCl, 1.36 mM CaCl₂ ⋅ 2H₂O and 2.38 mM NaHCO₃, 381 mOsm/kg. Undiluted sperm was used as the control. At 6 h poststorage, there were no substantial changes in spermatozoa motility and velocity at 30 s postactivation in all groups. Over 48 h of storage, the highest spermatozoa motility and velocity were obtained in sperm diluted in IM2 compared to the other groups. IM2 could maintain a significantly higher ATP content of diluted sperm than IM1 and undiluted treatment for 2 days. Similarly, the highest values of eyeing and hatching rates were observed in sperm diluted in IM2 compared to sperm in the other studied groups. It can be concluded that the obtained result is a novel and applicable approach to maintain semen quality of pikeperch during short-term storage, suggesting IM2 as a promising medium for short-term storage. The present study also opens possibilities for ensuring a reliable source of semen as a convenient approach for increasing genetic diversity in hatcheries.     

Artificial fertilization of gametes from the South African clawed frog,Xenopus laevis

A reproducible and effective method for fertilization eggs of Xenopus laevis was developed based of systematic manipulation of environmental factors. The effects of varying concentrations of individual components of a fertilization medium were tested by measuring jelly swelling, sperm motility, and sperm longevity. Results were used to develop an improved medium for fertilization, consisting of 41.25 mM NaCl, 1.25 mM KCl, 0.25 mM CaCl₂, 0.0625 mM MgCl₂, 0.5 mM Na₂HPO₄, 2.5 mM HEPES, 1.9 mM NaOH, final pH(2°) 7.8.     

Incubation of mouse sperm with lactate delays capacitation and hyperactivation and lowers fertilization levels in vitro

Mouse sperm were incubated in medium with or without 24 mM lactate and assessed for (1) motility characteristics including hyperactivation—a computer-assisted motion analysis system was used; (2) capacitation—a chlortetracycline fluorescent dye binding assay was used; and (3) ability to penetrate oocytes. Lactate affected all aspects of motility and delayed the rates of both hyperactivation and capacitation. When a concentration of 8 × 10³ sperm/ml was used for insemination in vitro, sperm preincubated 60–90 minutes in medium with lactate prior to insemination in lactate-free medium fertilized fewer oocytes than did sperm preincubated in lactate-free medium. Use of a calcium-sensitive electrode demonstrated that lactate chelated appreciable amounts of calcium in the medium. Capacitation was assayed in sperm incubated 60 minutes in medium with various concentrations of lactate or CaCl₂. When medium containing lactate was compared to medium without lactate but having a similar level of free calcium, the level of capacitation of sperm incubated with lactate was less than half that of sperm incubated without lactate. These results demonstrate that including 24 mM lactate in the medium can have detrimental effects on mouse sperm hyperactivation and capacitation. The detrimental effects on capacitation are partly but not completely due to the chelation of calcium by lactate.     

Protective effect of taurine,glutathione and trehalose on the liquid storage of ram semen

Oxidative damage to sperm resulting from reactive oxygen species generated by the cellular components of semen during liquid storage is possibly one of the main causes for the decline in motility and fertility during storage—the other detrimental cause is low temperature on the destabilisation of sperm membrane structure. The aim of this study was to determine the effects of the addition of the anti-oxidants taurine and glutathione (GSH), and the membrane structure stabiliser, trehalose, on sperm viability during low temperature liquid storage. A total number of 36 ejaculates were collected using the artificial vagina from four Chios rams and nine replicates of the ejaculates were diluted with a Tris-based extender containing additives as the control. The sperm motility, percentage abnormal sperm, plasma membrane intact sperm and the hypo-osmotic swelling test (HOST) were determined during storage of semen at 5 °C for a period of 0, 6, 24 and 30 h of liquid storage, respectively. Trehalose at a level of 50 mM provided the best maintenance of motility at 6 and 30 h (P < 0.05), and gave the highest percentage (69.0 ± 2.0% and 64.6 ± 1.8%, respectively) of viable sperm at 24 and 30 h (P < 0.01). Trehalose treatment at a concentration of 50 mM also resulted in the highest percentage of membrane-intact sperm (53.7 ± 2.9%) after performing HOST at 30 h. The anti-oxidant treatments GSH 5–10 mM and taurine at 50 mM provided a significant improvement in sperm survival during the 6 h of liquid storage at 5 °C (P < 0.05). In conclusion, many aspects of sperm protection, e.g. sperm motility, viability and membrane stabilisation of the sperm cells during relative low temperature storage, are the key factors determining the preservation of sperm function. Future efforts toward improving function of ram sperm kept in low temperature storage should concentrate on anti-oxidant additives. The results of this study provide a new approach to the preservation of sperm from rams of the Chios and related breeds, and so contribute to the improvement of these breeds for the world sheep industry.     

Successful ultrarapid cryopreservation of wild Iberian ibex (Capra pyrenaica) spermatozoa

A method for cryopreserving wild ibex sperm at high cooling rates was developed. To design a freezing solution based on Tris, citric acid, and glucose (TCG), two preliminary experiments were performed using glycerol (GLY) and dimethyl sulfoxide (DMSO) at different concentrations (5%, 10%, 20%). The 10% GLY + 10% DMSO combination reduced (P < 0.05) frozen-thawed sperm motility, which reached a minimum when 20% GLY + 20% DMSO was used. In the second experiment, sperm tolerance to three sucrose concentrations was evaluated (100-mM sucrose, 300-mM sucrose, 500-mM sucrose). Frozen-thawed sperm motility and sperm viability decreased (P < 0.05) at concentrations above 300 mM. The ultrarapid cooling procedure finally used involved a TCG egg yolk (ey)–based extender with 100-mM sucrose, either alone or with 5% GLY with or without BSA. Two warming procedures (37 °C vs. 60 °C) were also evaluated. The TCG ey with 100-mM sucrose but without GLY/BSA returned the best sperm quality variables. Slow warming at 37 °C strongly affected (P < 0.05) sperm motility and viability in all groups. Sperm selection by density gradient centrifugation produced no motile sperm when slow warming was performed. In contrast, when fast warming was used, sperm selection increased (P < 0.05) percentage of motility, viability, and the percentage of sperms with intact acrosomes. Heterologous in vivo fertilization involving domestic goats was performed to evaluate the in vivo fertilization capacity of the ultrarapidly cooled cryopreserved sperm (in TCG-ey + 100 mM sucrose), with warming undertaken at 60 °C. Inseminations of domestic goats resulted in three pregnancies (3 of 16, 18.7% fertility). In conclusion, ibex spermatozoa are strongly sensitive to high concentrations of permeable cryoprotectants and sucrose. However, the combination of ultrarapid cooling, using TCG-ey + 100-mM sucrose, and fast warming at 60 °C, followed by sperm selection by density gradient centrifugation to collect the motile sperm, has a positive effect on sperm viability.     

Comparison of the effects of glutamine and an amino acid solution on post-thawed ram sperm parameters,lipid peroxidation and anti-oxidant activities

Ram semen contains sufficient quantities of superoxide dismutase (SOD) and much lower concentrations of glutathione peroxidase (GSH-PX) and catalase (CAT) to prevent oxidative damage. The anti-oxidant capacity of the sperm cell is limited, due to a small cytoplasmic component, which contains these anti-oxidants to scavenge the oxidants. However, the concentration of these anti-oxidants may decrease considerably by the dilution of the semen. The aim of the present work was to study the effect of two anti-oxidants, namely, glutamine and an amino acid solution (BME) in a Tris-based extender on ram sperm parameters, lipid peroxidation and anti-oxidant capacity after the cryopreservation/thawing process. Ejaculates collected from 4 Akkaraman rams were evaluated and pooled at 37 °C. Semen samples which were diluted with the tris-based extender containing glutamine (2.5 or 5 mM), BME (13 or 26%), and no anti-oxidants (control) were cooled to 5 °C and frozen in 0.25-ml French straws and stored in liquid nitrogen. Frozen straws were thawed individually at 37 °C for 20 s in a water bath for evaluation. The freezing extender supplemented with 5 mM glutamine led to higher motility rate (68.0 ± 4.4%) and hypo-osmotic swelling test (HOST) (64.1 ± 5.5%), when compared to glutamine (2.5 mM) and BME (13 and 26%) (P < 0.05). No significant differences were observed regarding sperm motility and HOST, following the supplementation of the freezing extender with glutamine 2.5 mM and BME (13 and 26%) after thawing. CAT activity remained significantly higher following the addition of glutamine 5 mM (6.4 ± 0.9 kU/g protein), compared to the other treatments (P < 0.01). The anti-oxidants at different levels were not effective in the elimination of malondialdehyde (MDA) formation and maintenance of SOD activities, when compared to the control (P < 0.05). Findings showed that glutamine (5 mM) supplementation in semen extenders, was of greater benefit to frozen–thawed ram sperm. Future efforts are needed to find the appropriate anti-oxidants and their effective concentrations to improve post-thaw sperm parameters (e.g. motility, membrane integrity, fertility) and anti-oxidant activities when frozen–thawed ram sperm is used.     

Effects of supplementation of tris-egg yolk extender with different sugars and antioxidants on freezability of ram semen

This study was designed to determine the effects of the combined addition of different levels of certain sugars (trehalose, sucrose and raffinose) and antioxidants (vitamin E, C and taurine), in Tris-egg yolk extender on frozen-thawed ram semen parameters. Semen samples were collected from five healthy, mature and fertile Iranian Afshari rams, twice a week for 8 weeks. Selected samples were pooled and diluted with a Tris-egg yolk extender containing different levels of sugars and antioxidants. In Experiment 1, different levels of trehalose (0, 50 and 100 mM) were tested with different levels of taurine (0, 25 and 50 mM), vitamin E and C (0, 1 and 2 mM). In Experiment 2, different levels of sucrose (0, 60 and 80 mM) were tested with different levels of taurine (0, 25 and 50 mM), vitamin E and C (0, 1 and 2 mM). In Experiment 3, different levels of raffinose (0, 5, 10 mM) were tested with different levels of taurine (0, 25 and 50 mM), and vitamin E and C (0, 1 and 2 mM). In Experiment 4, the selected extenders of experiments 1, 2 and 3 were compared statistically with control (no selected sugar and antioxidant) extender. The results of experiments 1, 2 and 3 revealed that the highest frozen–thawed sperm parameters were recorded for the selected extenders containing 100 mM trehalose +2 mM vitamin E (T100E2), 60 mM sucrose + 2 mM vitamin E (S60E2) and 10 mM raffinose + 2 mM vitamin E (R10E2), respectively. The results of experiment 4 revealed that the post-thaw sperm total motility in T100E2 (62.41 ± 2.41%), S60E2 (59.52 ± 1.91%) and R10E2 (58.33 ± 2.00%) was higher than that of the control extender (46.00 ± 1.79%; P ≤ 0.05). Similarly, the progressive sperm motility in T100E2 (57.18 ± 1.96%), S60E2 (57.49 ± 1.94%) and R10E2 (55.03 ± 2.99%) was also higher than that of the control extender (41.20 ± 1.70%; P ≤ 0.05). Post-thaw sperm viability in selected extenders of T100E2 (65.20 ± 2.67%), S60E2 (62.00 ± 2.07%) and R10E2 (61.80 ± 2.46%) was higher than that of control extender (51.00 ± 1.88%; P ≤ 0.05). In conclusion, the addition of 100 mM trehalose, 60 mM sucrose and 10 mM raffinose combined with 2 mM vitamin E in Tris-egg yolk extender significantly improved frozen-thawed ram semen parameters.     

Cryopreservation of rainbow trout Oncorhynchus mykiss spermatozoa: Effects of extender supplemented with different antioxidants on sperm motility,velocity and fertility

In present study, it was examined whether addition of different antioxidants to the cryopreservation extenders had an effect on semen post-thaw fertility and motility in rainbow trout (Oncorhynchus mykiss) and also it was investigated the sperm characteristics post-thaw sperm characteristics and fertility. The collected semen was pooled to minimize individual variation. Each pooled ejaculate was split into 12 equal aliquots and diluted with base extenders supplemented with the antioxidants, and a base extender with no additives (control). The pooled semen samples diluted at the ratio of 1:10 by the extenders were subjected to cryopreservation. Antioxidants were separately added to the extenders (one per experimental group): catalase (250 U/l), superoxide dismutase (250 U/l), peroxidase (250 U/l), oxidized glutathione (1.5 mmol/l), reduced glutathione (1.5 mmol/l), l-methionine (1.5 mmol/l), uric acid (0.25 mmol/l), l-ascorbic acid (0.5 mmol/l), α-tocopherol (2.0 mmol/l), β-carotene (0.5 mmol/l) and carnitine (0.5 mmol/l). After dilution the semen was aspirated into 0.25 ml straws, the straws were placed on the tray, frozen for 10 min, and plunged into liquid nitrogen. Our results indicated that the post-thaw motility rate increased in extenders supplemented with uric acid, l-methionine, SOD, l-carnitine, α-tocopherol and l-reduced glutathione (p < 0.05). The motility duration of frozen thawed semen increased in extenders supplemented with uric acid, l-methionine, SOD, α-tocopherol and l-reduced glutathione (p < 0.05). Fertilization rate and hatching rate of frozen-thawed semen was not affected by the tested antioxidants. Consequently, the tested antioxidants affected the motility parameters and cryopreservation extenders could be supplement with antioxidants. This study suggested usage of antioxidants in the cryopreservation of rainbow trout.     

Optimization of the cryopreservation of African clawed frog (Xenopus laevis) sperm

Cryopreservation of testicular sperm in the African clawed frog, Xenopus laevis, was tested using three penetrating cryoprotectants (DMSO, methanol, and glycerol) and three semen diluents (300 mmol/L glucose, 300 mmol/L sucrose, and a motility inhibiting saline [MIS] solution [150 mmol/L NaCl, 3 mmol/L KCL, 1 mmol/L Mg₂SO₄, 1 mmol/L CaCl₂, and 20 mmol/L Tris, pH 8.0]). Three freezing rates and four thawing rates were also tested, and the best freezing/thawing conditions have been determined. The responses of sperm motility, viability, and fertility were assessed. Incubation of the sperm macerates with penetrating cryoprotectants showed that DMSO was the least toxic and methanol the most toxic. Semen in cryodiluents frozen 10 cm above the surface of liquid nitrogen (freezing rate of 20 to 25 °C/min) and thawed at room temperature for 40 sec had significantly higher percentages of motile and viable sperm than that of semen frozen 5 cm or 8 cm above the surface of liquid nitrogen and thawed at 5, 25, or 30 °C for 10, 15, or 60 sec, respectively. Sperm frozen in MIS containing 5% DMSO had a higher hatching rate than that of sperm frozen in sucrose and glucose diluents containing 5% or 10% DMSO and in MIS containing 10% DMSO. Addition of 73 mmol/L sucrose to the sperm extender MIS + 5% DMSO could improve the postthaw sperm motility and fertility. In conclusion, dilution of collected sperm in MIS solution (to have a final concentration of 6.5 × 10⁶ to 8 × 10⁶/mL) containing 5% DMSO and 73 mmol/L sucrose, freezing in a vapor of liquid nitrogen at 10 cm above the surface, and thawing at room temperature for 40 sec was the best cryopreservation protocol. This protocol gave 70% hatching rate, 80% motility rate, and 75% viability rate of fresh hormonally induced sperm.     

The addition of reduced glutathione to cryopreservation media induces changes in the structure of motile subpopulations of frozen-thawed boar sperm

Adding cryopreservation media with reduced glutathione (GSH) has previously been shown to maintain the motility, membrane integrity and fertilizing ability of frozen-thawed boar sperm, although the effects of GSH on good (GFE) and poor freezability (PFE) ejaculates rely upon the intrinsic ejaculate freezability. The resilience to withstand freeze-thawing procedures has previously been related to the existence of a specific distribution of motile sperm subpopulations, which differs between GFE and PFE. Thus, the main aim of this study was to determine whether the addition of GSH to freezing media has any impact on the distribution of motile sperm subpopulations in GFE and PFE. With this purpose, 18 GFE and 13 PFE were cryopreserved with or without 2 mM GSH. Sperm quality and motile subpopulations were evaluated at 30 min and 4 h post-thawing. Three subpopulations were identified and the percentages of spermatozoa belonging to the fastest and most linear subpopulation, which was referred as ‘SP1’, decreased over post-thawing time. Good freezability ejaculates that were cryopreserved in the presence of 2 mM exhibited a significantly higher percentage of spermatozoa belonging to SP1 than the other combinations of treatment and freezability both at 30 min (mean ± SEM: GFE-C: 16.6 ± 0.4; GFE-GSH 27.7 ± 0.6) and 4 h post-thawing (GFE-C: 7.8 ± 0.2 vs. GFE-GSH: 16.7 ± 0.4). In conclusion, the positive effect of GSH on the motility of frozen-thawed sperm is related to a specific sperm subpopulation (SP1), which could coincide with the fertile sperm one.     

Effects of antioxidants on post-thawed bovine sperm and oxidative stress parameters: Antioxidants protect DNA integrity against cryodamage

This study was conducted to determine the effects of methionine, inositol and carnitine on sperm (motility, abnormality, DNA integrity and in vivo fertility) and oxidative stress parameters (lipid peroxidation, total glutathione and antioxidant potential levels) of bovine semen after the freeze–thawing process. Nine ejaculates, collected with the aid of an artificial vagina twice a week from each Simmental bovine, were included in the study. Each ejaculate, splitted into seven equal groups and diluted in Tris-based extender containing methionine (2.5 and 7.5 mM), carnitine (2.5 and 7.5 mM), inositol (2.5 and 7.5 mM) and no additive (control), was cooled to 5 °C and then frozen in 0.25 ml straws. Frozen straws were then thawed individually at 37 °C for 20 s in a water bath for the evaluation.The extender supplemented with 7.5 mM doses of carnitine and inositol led to higher subjective motility percentages (61.9 ± 1.3% and 51.3 ± 1.6%) compared to the other groups. The addition of methionine and carnitine at doses of 2.5 and 7.5 mM and inositol at doses of 7.5 mM provided a greater protective effect in the percentages of total abnormality in comparison to the control and inositol 2.5 mM (P < 0.001). As regards CASA motility, 7.5 mM carnitine (41.6 ± 2.9% and 54.2 ± 4.9%) and inositol (34.9 ± 2.0% and 47.3 ± 2.2%) caused insignificant increases in CASA and total motility in comparison to the other groups. All of the antioxidants at 2.5 and 7.5 mM resulted in lower sperm with damaged DNA than that of control, thus reducing the DNA damage (P < 0.05). No significant differences were observed in CASA progressive motility and sperm motion characteristics among the groups. In fertility results based on 59-day non-returns, no significant differences were observed in non-return rates among groups. As regards biochemical parameters, supplementation with antioxidants did not significantly affect LPO and total GSH levels in comparison to the control group (P > 0.05). The maintenance of AOP level in methionine 2.5 mM was demonstrated to be higher (5.06 ± 0.38 mM) than that of control (0.96 ± 0.29 mM) following the freeze–thawing (P < 0.001). Supplementation with these antioxidants prior to the cryopreservation process protected the DNA integrity against the cryodamage. Furthermore, future research should focus on the molecular mechanisms of the antioxidative effects of the antioxidants methionine, carnitine and inositol during cryopreservation.     

Successful sperm cryopreservation of the brown-marbled grouper,Epinephelus fuscoguttatus using propylene glycol as cryoprotectant

This study developed the cryopreservation of brown-marbled grouper spermatozoa for practical application. We examined 32 cryodiluents, developed from four types of cryoprotectants [propylene glycol (PG), dimethyl-sulphoxide (Me₂SO), dimethyl-acetamide (DMA) and ethylene glycol (EG)] at four concentrations of 5, 10, 15 and 20% in combination with two extenders [Fetal bovine serum (FBS) and artificial seminal plasma (ASP). Cooling rates were examined by adjusting the height of straws (2.5–12.5 cm) from the liquid nitrogen (LN) vapor and cooled for 5 min before immersion into LN. DNA laddering was used to detect DNA damage in cryopreserved sperm. In fertilization trials, 0.5 g of eggs was mixed with cryopreserved sperm stored for 30 days in LN. The best motility of post-thaw sperm was achieved using 15% PG + 85% FBS (76.7 ± 8.8%); 10% PG + 90% FBS was also effective as cryodiluent. Generally, FBS gave better post-thaw motility compared to ASP. The optimum cooling rate was at 17.6 °C min⁻¹ obtained by freezing at the height of 7.5 cm surface of LN. The results obtained showed that cryopreserved sperm of brown-marbled grouper suffered slight DNA fragmentation, which resulted in significantly lower motility. However, the fertilization (90.9 ± 0.5%), hatching (64.5 ± 4.1%) and deformity rates (3.8 ± 0.2%) obtained from cryopreserved sperm showed no significant difference with fresh sperm. These findings show that the developed protocol for cryopreservation of brown-marbled grouper sperm was viable and will be useful for successful breeding and seed production of brown-marbled grouper.     

Effects of antioxidants on post-thawed bovine sperm and oxidative stress parameters: Antioxidants protect DNA integrity against cryodamage

This study was conducted to determine the effects of methionine, inositol and carnitine on sperm (motility, abnormality, DNA integrity and in vivo fertility) and oxidative stress parameters (lipid peroxidation, total glutathione and antioxidant potential levels) of bovine semen after the freeze–thawing process. Nine ejaculates, collected with the aid of an artificial vagina twice a week from each Simmental bovine, were included in the study. Each ejaculate, splitted into seven equal groups and diluted in Tris-based extender containing methionine (2.5 and 7.5 mM), carnitine (2.5 and 7.5 mM), inositol (2.5 and 7.5 mM) and no additive (control), was cooled to 5 °C and then frozen in 0.25 ml straws. Frozen straws were then thawed individually at 37 °C for 20 s in a water bath for the evaluation.The extender supplemented with 7.5 mM doses of carnitine and inositol led to higher subjective motility percentages (61.9 ± 1.3% and 51.3 ± 1.6%) compared to the other groups. The addition of methionine and carnitine at doses of 2.5 and 7.5 mM and inositol at doses of 7.5 mM provided a greater protective effect in the percentages of total abnormality in comparison to the control and inositol 2.5 mM (P < 0.001). As regards CASA motility, 7.5 mM carnitine (41.6 ± 2.9% and 54.2 ± 4.9%) and inositol (34.9 ± 2.0% and 47.3 ± 2.2%) caused insignificant increases in CASA and total motility in comparison to the other groups. All of the antioxidants at 2.5 and 7.5 mM resulted in lower sperm with damaged DNA than that of control, thus reducing the DNA damage (P < 0.05). No significant differences were observed in CASA progressive motility and sperm motion characteristics among the groups. In fertility results based on 59-day non-returns, no significant differences were observed in non-return rates among groups. As regards biochemical parameters, supplementation with antioxidants did not significantly affect LPO and total GSH levels in comparison to the control group (P > 0.05). The maintenance of AOP level in methionine 2.5 mM was demonstrated to be higher (5.06 ± 0.38 mM) than that of control (0.96 ± 0.29 mM) following the freeze–thawing (P < 0.001). Supplementation with these antioxidants prior to the cryopreservation process protected the DNA integrity against the cryodamage. Furthermore, future research should focus on the molecular mechanisms of the antioxidative effects of the antioxidants methionine, carnitine and inositol during cryopreservation.     

Effects of ovarian fluid on motility characteristics of rainbow trout (Oncorhynchus mykiss Walbaum) spermatozoa

The objective of this work was to determine sperm motility characteristics in rainbow trout ( Oncorhynchus mykiss Walbaum) (nine males) and assess the motility enhancement potential of ovarian fluid of nine females in order to test gender effects on sperm motion. Spermatozoa motility characteristics were obtained using computer-assisted sperm analysis (CASA). Ovarian fluid enhanced sperm motility and this was not attributed to the percentage of motile sperm in the samples but rather to the speed and trajectory of movement. For example straight-line velocity (VSL) increased from 32.3 to 85.8 μm s⁻¹ and amplitude of lateral head displacement (ALH) decreased from 20.11 to 10.25 μm. Some females produced ovarian fluid with clearly different sperm motility-enhancement capacity. Furthermore, spermatozoa of some males performed differently regardless of the origin of the ovarian fluid. These data indirectly suggest the existence of cryptic female choice in rainbow trout.     

Characterization of lake minnow Eupallasella percnurus semen in relation to sperm morphology,regulation of sperm motility and interpopulation diversity

Sperm morphology and regulation of sperm motility of lake minnow Eupallasella percnurus, an endangered cyprinid, were investigated. Milt characteristics from two isolated populations of E. percnurus were compared to characterize the interpopulation diversity. Electron microscopic studies revealed that E. percnurus spermatozoa comprise simple, uniflagellate, anacrosomal aquasperm with species‐specific features as an eccentrically located implantation of nuclear fossa and eccentric insertion of flagellum. Sperm motility was significantly inhibited by relatively low ion concentrations (150, 150 and 8 mM for NaCl, KCl and CaCl₂, respectively). Sperm maintained a high motility rate over a wide pH range (5·5–10·5), which may reflect adaptation to a highly variable environment. The two E. percnurus populations were markedly different in milt volume, sperm concentration, seminal plasma pH, sperm motility and beat cross frequency, which may result from genetic differences and environmental conditions.