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.     

Effect of substituting different concentrations of soybean lecithin and egg yolk in tris-based extender on goat semen cryopreservation

This study aimed to investigate the effects of different concentrations of soybean lecithin (SL; 0.5%, 1%, and 1.5%) and egg yolk (EY) in Tris-based extenders on the semen quality parameters of post-thawed goat semen. Sixteen ejaculates were collected from eight healthy, mature Chongming White goats (3–5 years of age). Each ejaculate was divided into five equal aliquots, and then each pellet was diluted with one of the five Tris-based extenders containing 20% EY, 0.5% SL, 1% SL, 2% SL, or 3% SL. The cooled diluted semen was loaded into 0.5 mL polyvinyl French straws and cryopreserved in liquid nitrogen. Frozen semen samples were thawed at 37 °C and assessed for sperm motility, viability, plasma acrosome integrity, membrane integrity, and mitochondria integrity, and the spermatozoa were assessed for reactive oxygen species (ROS), superoxide dismutase (SOD), and malondialdehyde (MDA). The semen extended in the 2.0% SL extract tended to have a higher sperm viability (57.44%), motility (52.14%), membrane integrity (45.31%), acrosome integrity (52.96%), and mitochondrial activity (50.21%) than the other SL-based extender concentrations (P < 0.05). The 2.0% SL treatment group was equivalent to the semen extended in 20% EY (P > 0.05). The extenders supplemented 20% EY or 2.0% SL significantly increased the SOD activity and decreased the ROS and MDA activities compared to the other groups (P < 0.05). In conclusion, the extenders supplemented with 20% EY and 2.0% SL had similar effects on spermatozoa preservation. These results indicate that a soybean lecithin-based diluent may be used as an alternative extender to egg yolk for the cryopreservation of goat semen.     

Royal jelly supplemented soybean lecithin-based extenders improve post-thaw quality and incubation resilience of goat spermatozoa

The aim of the present study was to evaluate different concentrations of royal jelly (RJ) supplemented extenders for post-thaw quality and incubation resilience of goat spermatozoa. Semen samples were collected from five goats. Pooled semen were diluted with soybean lecithin-based extender without RJ (control) or supplemented with different concentrations (0.25, 0.5 and 0.75%) of RJ (RJ0.25, RJ0.5, RJ0.75 respectively), at a final concentration of 150 × 10⁶ spermatozoon/mL. Semen samples were assessed for sperm motility, plasma membrane 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).The addition of RJ (0.5%, 0.75%) led to higher percentages of subjective motilities (55.33 ± 2.29%, 57.67 ± 2.58%) compared to control and RJ0.25 groups (49.00 ± 2,80%, 51.67 ± 3.09%) (P < 0.05) following the freeze–thawing process. RJ0.5 and RJ0.75 groups had higher plasma membrane functional integrities (66.40 ± 1.34%, 68.20 ± 2.05%) and lower defected acrosome rates (24.60 ± 3.36%, 23.80 ± 2.27%) compared to the other groups (P < 0.05). DNA damaged spermatozoa in all groups were not significant (P > 0.05).In the end of incubation, motility and HOST rates of RJ0.5 (14.00 ± 3.87%, 31.20 ± 3.70%) and RJ0.75 (15.00 ± 3.27%, 29.20 ± 2.59%) groups were higher than control (8.00 ± 2.54%, 18.20 ± 3.11%) and RJ0.25 (9.00 ± 2.07%, 20.60 ± 2.88%) groups (P < 0.05). Also defected acrosome and DNA fragmation rates of RJ0.5 (32.20 ± 1.30%, 5.4 ± 0.55%) and RJ0.75 (29.20 ± 1.30%, 5.80 ± 0.45%) groups were significantly lower than control (38.80 ± 0.84%, 7.40 ± 1.34%) and RJ0.25 (39.80 ± 2.05%, 7.00 ± 1.58) groups. This study shows that RJ supplemented extenders have beneficial effect on goat sperm parameters at 0 h and 6 h of incubation.     

Sperm quality of Colossoma macropomum after room‐temperature and cold storage

The objective of this study was to evaluate the effects of cold and room‐temperature storage on the quality of Colossoma macropomum sperm. The experiment was carried out in December (end of Spring), in Nova Mutum‐MT, Brazil, involving nine C. macropomum males (4 years old; 6.4 ± 1.5 kg average weight). The fish were selected and transferred to masonry tanks (4 m³) in a laboratory (water renewal rate: 10 L/s; average water temperature: 28°C). Subsequently, reproduction was induced using 2.5 mg of crude carp pituitary extract/kg and the semen was harvested 240 degree hours after hormonal induction. The following sperm characteristics were analyzed every 5 hr using Image J/casa software: total motility (MOT), curvilinear velocity (VCL), average path velocity (VAP), straight‐line velocity (VSL), straightness of sperm path (STR), wobble (WOB), progressive motility (PROG), beat cross frequency (BCF) and total number of spermatozoa (NSPZ). A fresh sample of semen from each animal was kept at room temperature (25.3 ± 1.2°C). For analysis of cooled semen, syringes were kept in cooling boxes at an average temperature of 16.9 ± 2.1°C. The reduction (p < 0.05) of MOT in semen kept at room temperature occurred at 10 hr (13.95%); in cooled semen, however, MOT declined at 15 hr (76.87%). At 15 hr, there was practically no MOT in the semen kept at room temperature (0.20%), whereas in the cooled semen this situation was observed only at 35 hr (2.91%) The MOT of cooled sperm was higher (p < 0.05) at all times (except zero time), compared with the semen maintained at room temperature. At 15 hr, the cooled spermatozoa showed higher (p < 0.05) VCL (142.18 μm/s) and BCF (29.72 Hz) than those maintained at room temperature (VCL: 51.18 μm/s; BCF: 19.57 Hz). After 15 hr, only the cooled sperm showed quality. In conclusion, semen cooling allows for extending the viability of C. macropomum spermatozoa from 5 to 10 hr without compromising their quality in most characteristics. At 15 and 25 hr of cooling, sperm viability is still observed, though with decreased quality.     

Cryopreservation of giant grouper Epinephelus lanceolatus (Bloch, 1790) sperm

The grouper, Epinephelus lanceolatus, is a vulnerable species of high economic value. An effective protocol was developed for the cryopreservation of E. lanceolatus by comparing different extenders produced by mixing various cryoprotectants (dimethyl sulfoxide, methanol and glycerol) and diluents (MPRS, TS‐2, TS‐19, Cortland and Hank's). Using computer‐assisted sperm analysis (CASA) and morphological analysis, the sperm motility and fertilization rates from post‐thaw sperm were comparable to untreated controls. The results revealed that MPRS (containing 12% DMSO) or TS‐19 (containing 12% DMSO), were the optimum extenders for protecting the sperm from cryo‐damage in liquid nitrogen. The post‐thaw sperm maintained high motility (90.61 ± 3.03%) and a fertilization rate (92.27 ± 2.43%) similar (P > 0.05) to fresh sperm (94.34 ± 4% and 94.10 ± 1.87%). This study is the first to report on the successful sperm cryopreservation of E. lanceolatus and provides an important tool for repopulating this species through aquaculture.     

Effects of mint,thyme, and curcumin extract nanoformulations on the sperm quality,apoptosis, chromatin decondensation,enzyme activity,and oxidative status of cryopreserved goat semen

Goat semen cryopreservation is a challenging process as it results in reduced motility, vitality, and fertility of spermatozoa after freezing. In this study, we evaluated the effects of different herbal extract nanoformulations (NFs) [mint (MENFs), thyme (TENFs), and curcumin (CENFs)], supplemented at either 50 or 100 μg into Tris-extender on the cryopreserved goat semen quality. The hydrothermal squeezing method was used for the preparation of the NFs extracts. The morphological evaluation of the NFs extracts was conducted by transmission electron microscopy. All NFs supplements improved (p < 0.05) the progressive motility, vitality, and plasma membrane integrity of sperm compared with the control extender after equilibration (5 °C for 2 h) and thawing (37 °C for 30 s), but had no effect on sperm abnormality and acrosome integrity. All NFs supplements decreased (p < 0.05) the apoptosis, malondialdehyde level, and chromatin decondensation of sperm cells, while increased (p < 0.05) the total antioxidant capacity and catalase activity in the frozen/thawed extender. Particularly, CENFs at a level of 100 μg showed improvement of sperm parameters and antioxidant status during cryopreservation of goat semen more than TENFs and MENFs. The CENFs improved the quality of goat spermatozoa in post-thawed semen in terms of preventing cryodamage and promoting the cryotolerance of spermatozoa when compared with TENFs and MENFs. Therefore, supplementation of Tris-extender with CENFs could enhance goat semen processing during cryopreservation.     

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.     

The role of major histocompatibility complex molecules in luteal function

Background

Turkey reproduction is by artificial insemination using pooled semen so there is interest in storing semen. Fertilizing capacity declines after six hours storage, possibly due to poor sperm mobility. Prostaglandins (PG) affect mammalian sperm motility, but avian sperm has not been widely studied. For this study, levels of PG E1, E2, and F2 alpha in turkey seminal plasma and sperm extract, and effects of cyclooxygenase (COX) inhibitors on sperm mobility were determined.

Methods

Seminal Plasma and sperm extract PG E1, E2, and F2 alpha, from 1.0 mL pooled semen, were measured by ELISA. In Trial 1, PG were determined from 122 wk old toms (n = 4). Trial 2 used 36 wk old toms (n = 7). For Trial 3, PGE2 only was measured from 48 wk (n = 6) and 154 wk old toms (n = 3). The effects of non-specific COX inhibitors indomethacin, diclofenac, tolmetin, or aspirin (n = 10), or specific COX-1 or COX-2 inhibitors (n = 3) on sperm mobility were measured (Accudenz swim-down test).

Results

Seminal plasma PG (pg/mL) in Trials 1 and 2, respectively, were 185.2 ± 88.4 and 187.2 ± 33.7 for PGE1; 141.4 ± 43.1 and 100.4 ± 14.6 for PGF2 alpha; and 431.0 ± 155.1 for PGE2 (Trial 1 only). Sperm extract PG (pg/10 billion cells) in Trials 1 and 2, respectively, were 215.1 ± 38.1 and 208.9 ± 41.5 for PGE1; 133.7 ± 51.7 and 49.8 ± 8.3 for PGF2 alpha; and 52.3 ± 8.6 for PGE2 (Trial 1 only). In Trial 3, seminal plasma PGE2 (pg/mL) in older versus younger males was 1097.9 ± 99.3 versus 853.2 ± 144.6 and sperm extract PGE2 (pg/10 billion cells) was 208.0 ± 56.1 versus 102.4 ± 14.8. Cyclooxygenase inhibitors (0.001 to 10 mM) decreased sperm mobility: indomethacin 15 to 100%; diclofenac 4 to 100%; tolmetin 27 to 74%; aspirin (tested at 0.01 to 15 mM) 22 to 42%; resveratrol (COX-1) and NS-398 (COX-2), both tested at 0.1 to 10 mM, 38 to 98% and 44 to 85%, respectively.

Conclusion

These results indicate that PG are present in turkey seminal plasma and sperm, and COX inhibitors decrease turkey sperm mobility.     

The breeding management affects fresh and cryopreserved semen characteristics in Melopsittacus undulatus

Melopsittacus undulatus is a companion parrot worldwide diffused. Many parrots are considered endangered or vulnerable. The preservation of semen is crucial in endangered species, thus, M. undulatus could be a good model to study sperm characteristics and semen cryopreservation in these other endangered parrots. In this study the effect of the breeding management (males bred in promiscuous aviary or in couple) on sperm characteristics (motility, membrane integrity and morphometry) of fresh and cryopreserved semen was evaluated. The computer-assisted sperm analysis (CASA) revealed a significant effect of the husbandry method on semen characteristics in budgerigars: male housed in couple with the female in individual cages allowed the higher results in term of both semen quantity and sperm quality. Total and progressive motility were significantly higher in males bred in couple (68.7 ± 8.9% and 54 ± 15.9%, respectively) than in promiscuous aviary (48.3 ± 15.1% and 24.4 ± 12.4%, respectively), such as sperm velocity (average path velocity, straight line velocity, and curvilinear velocity). The type of sperm movement (amplitude of lateral head displacement, beat cross frequency, straightness, and linearity), sperm membrane integrity and morphometry parameters seemed not affected by the husbandry method. The standardization of a CASA procedure for the semen analysis in M. undulatus allow further studies on parrot semen manipulation and cryopreservation, but the method used for the breeding of the male could have a significant effect on the semen quality.     

Impact of supplementation of semen extender with antioxidants on the quality of chilled or cryopreserved Arabian stallion spermatozoa

The aim of the present study was to evaluate the effects of supplementation of semen extender with various non-enzymatic antioxidants on the quality of cooled or cryopreserved Arabian stallion spermatozoa. Semen collected from four pure Arabian stallions was centrifuged at 600g for 15 min. Spermatozoa were then diluted in INRA-82 extender supplemented with bovine serum albumin (BSA; 0, 10, 15 and 20 mg/mL) or trehalose (0, 75, 100 and 150 mM) or zinc sulphate (0, 100, 150 and 200 μM). The diluted semen was then either cooled at 5 °C or cryopreserved in 0.5–ml plastic straws. After cooling or thawing, sperm motility, viability, sperm abnormalities, viability index, and plasma membrane integrity were evaluated. The results showed that supplementation of semen extender with 150 mM trehalose or with 200 μM zinc sulphate significantly (P < 0.05) improved motility, viability, sperm membrane integrity and acrosome status in Arabian stallion spermatozoa after cooling or after freezing and thawing compared with controls (non-supplemented media) or with those supplemented with other concentrations of trehalose or zinc sulphate. Supplementation of semen extender with BSA did not improve sperm motility or cryosurvival of Arabian stallion spermatozoa after cooling or after freezing and thawing. In conclusion, supplementation of semen extender with non-enzymatic antioxidants (trehalose or zinc sulphate) improved the quality of chilled and frozen/thawed Arabian stallion spermatozoa. The most beneficial effects occur when semen diluent was supplemented with 150 mM trehalose or 200 μM zinc sulphate.     

Dietary omega-3 fatty acids from linseed oil improve quality of post-thaw but not fresh sperm in Holstein bulls

Docosahexaenoic acid (DHA), a member of the n-3 fatty acid family present in fish oil, has several positive effects on bovine sperm, including membrane integrity, motility and viability, as well as cold sensitivity. Our objective was to investigate effects of varying amounts of omega-3 fatty acids from linseed oil, administered orally, on quality of fresh and frozen-thawed bull sperm. Twenty fertile Holstein bulls (874 ± 45.38 kg) were randomly and equally assigned to four groups and received encapsulated (rumen-protected) fats for 12 weeks, as follows: group P, 300 g palm oil; group Pl, 200 g palm oil + 100 g linseed oil; group pL, 100 g palm oil + 200 g linseed oil; and group L, 300 g linseed oil. Sperm quality of fresh and frozen-thawed semen was evaluated by routine assays including sperm motion characteristics (CASA), membrane integrity (eosin-nigrosin), membrane activity (hypo-osmotic swelling test; HOST) and malondialdehyde (MDA) content. There were no significant differences among groups in semen volume, sperm concentration or sperm quality parameters in fresh semen. However, after freezing-thawing, total and progressive motility in group P (59.61 ± 1.95 and 40.19 ± 2.48%, respectively; LSM ± SEM) were lower (P < 0.05) than in groups Pl (66.06 ± 1.95 and 47.53 ± 2.48%), pL (65.67 ± 1.95 and 47.48 ± 2.48%) and L (65.36 ± 1.95 and 47.62 ± 2.48)%, with no significant differences among the latter three groups. Furthermore, membrane integrity (eosin-nigrosin) and activity (HOST) were lower (P < 0.05) in group P (55.79 ± 2.15 and 42.19 ± 2.17%) compared to groups Pl (62.73 ± 2.15 and 48.93 ± 2.17%), pL (64.06 ± 2.15 and 50.01 ± 2.17%) and L (64.47 ± 2.15 and 49.68 ± 2.17%), with no significant differences among the latter three. Furthermore, there were more (P < 0.05) morphologically abnormal sperm in group P (25.99 ± 1.62%) than in groups Pl, PL and L (21.55 ± 1.62, 21.69 ± 1.62 and 20.90 ± 1.62%). In conclusion, feeding Holstein bulls 100–300 g linseed oil daily improved sperm cryotolerance.     

Effect of dilution rate on feline urethral sperm motility,viability, and DNA integrity

This study was designed to investigate if the characteristics of feline urethral sperm can be affected by high dilution in an artificial medium. The semen collected by urethral catheterization from eight male cats was evaluated for sperm concentration and motility and subsequently diluted with a TRIS-based extender to the concentration of spermatozoa 10 × 10⁶/mL, 5 × 10⁶/mL, and 1 × 10⁶/mL. Immediately after the extension samples were assessed for motility, cell viability using SYBR-14 and propidium iodide, acrosome integrity using lectin from Arachis hypogaea Alexa Fluor 488 Conjugate, and propidium iodide and chromatin status by acridine orange. Compared with 10 × 10⁶/mL dilution rate, spermatozoa diluted to 1 × 10⁶ sperm/mL had a significantly lower proportion of motile (31.1% ± 19.8 and 0.7% ± 1.6, respectively, P < 0.05) and viable spermatozoa (88.3% ± 3.1 and 69.1% ± 12.8, respectively, P < 0.01). There was no dilution-related difference in the acrosome integrity (76.7% ± 11.9 vs. 75.9% ± 10.6) and chromatin status (defragmentation index, 3.3% ± 0.97 vs. 3.4% ± 1.7). These results indicate that feline urethral semen is susceptible to high dilution rate, and some sperm characteristics can be artifactually changed by semen dilution. It also suggests the potential role of seminal plasma in maintaining sperm motility and viability in high dilution rates.     

Semen collection,characterization, and cryopreservation in a Magellanic penguin (Spheniscus magellanicus)

A cooperative method was developed for collecting semen from a Magellanic penguin. Ejaculate parameters and semen production during a breeding season were characterized. Experiments were performed to study the effect on penguin spermatozoa of two temperatures (4°C and 21°C) for short‐term storage, and two cryoprotectants (dimethylsulfoxide [DMSO] and ethylene glycol [EG]) for long‐term storage (cryopreservation). All dilutions were made using modified Beltsville Poultry Semen Extender. Sperm quality was assessed by evaluating motility and forward progression (sperm motility index [SMI]), viability, and morphology. A total of 39 ejaculates was collected over the 40‐day study period. Thirty‐eight ejaculates contained spermatozoa, but semen quality decreased toward the end of the study period. Varying levels of urate contamination were present in all ejaculates. Sperm quality parameters were similar for diluted samples held at 4°C and 21°C, and samples maintained high numbers of viable (77.8 ± 5.4%) and morphologically normal (67.9 ± 2.5%) spermatozoa at 3 hr. SMI and percentage of viable sperm decreased (P < 0.05) and the number of spermatozoa with a bent head or midpiece increased (P < 0.05) for both temperature groups over the 3‐hr storage interval. DMSO and EG were equally effective in maintaining penguin sperm quality parameters during the cryopreservation and thawing process. Frozen‐thawed semen maintained 69 ± 5 and 78 ± 3% of its pre‐freeze SMI and viability, respectively. SMI and viability decreased slightly during the cooling and equilibration phases but remained relatively stable during the 3‐hr storage interval post‐thaw. Frozen‐thawed semen also exhibited an increase (P < 0.05) in spermatozoa with a bent head or midpiece over time. The pre‐freeze SMI was higher (P < 0.05) for ejaculates with low levels of urates (clean ejaculates) compared with ejaculates with high levels of urate contamination, but sperm viability and morphology were similar (P > 0.05). Both SMI and viability of frozen‐thawed spermatozoa were higher (P < 0.05) for clean than for contaminated ejaculates. This is the first report on penguin ejaculate parameters, semen production, and preliminary methods for short‐ and long‐term semen storage. Zoo Biol 18:199–214, 1999. © 1999 Wiley‐Liss, Inc.     

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.     

Sperm cryostorage in a dry tank: An accurate alternative

This prospective study aimed to determine the effects of dry nitrogen cryostorage on human sperm characteristics in comparison with liquid nitrogen cryostorage. For this purpose, 42 men undergoing routine semen analysis (21 normozoospermia and 21 with altered semen parameters) were analyzed. After slow freezing, half of the straws of each sample were randomly stored in liquid and dry tanks, at the top and bottom levels of the latter. After 6 months storage, thawed samples were treated by density gradient centrifugation and sperm characteristics were compared. There was no difference in sperm progressive motility (15.1% ± 14.2% vs. 15.1% ± 12.7%; p = 0.76), sperm vitality (25.5% ± 17.7% vs. 26.2% ± 19%; p = 0.71), percentages of acrosome-reacted spermatozoa (38% ± 8.5% vs. 38.5% ± 7.4%; p = 0.53) and DNA fragmentation spermatozoa (27.3% ± 12.4% vs. 28.5% ± 12.9%, p = 0.47) after cryostorage in the dry or the liquid nitrogen tank. Moreover, we did not observe differences between either cryostorage system for normal and altered sperm samples. This lack of difference was also observed whatever the floor level of cryostorage in the dry tank. The temperature measurement of the dry tank showed a stable temperature at −194 °C throughout storage whatever the storage floor level, guaranteeing the stability of the low temperatures suitable for human sperm storage. Because of its greater safety, dry storage without contact with the liquid phase should be preferred and can be a useful alternative for the cryostorage of human sperm samples.     

Morphology, chemical contents and physiology of chondrostean fish sperm: a comparative study between Siberian sturgeon (Acipenser baerii) and sterlet (Acipenser ruthenus)

The present study was carried out with two sturgeon species, Siberian sturgeon (Acipenser baerii) and sterlet (A. ruthenus) to compare spermatological parameters to better understand inter‐species differences. Significant differences between morphometric features were observed such as acrosome length, acrosome width, head length, midpiece width and flagellar length, while midpiece length did not reveal such differences. The sterlet has a shorter spermatozoon than the Siberian sturgeon. Ultrastructural parameters vary significantly in terms of length of the nucleus, diameter of the endonuclear canals (EC), size of posterolateral projections (PP) and diameter of flagellum. Mean values for density of spermatozoa in the semen, seminal plasma pH, osmolality (mOsmol kg^?1), along with Ca²⁺, Na⁺, K⁺, Cl^? ions concentrations (mm ) were determined to be 0.61 ± 0.37 × 10⁹, 8.16 ± 0.18, 77.20 ± 52.28, 0.24 ± 0.06, 31.39 ± 10.21, 3.51 ± 1.10, 14.00 ± 4.30 in A. baerii and 0.41 ± 0.32 × 10⁹, 8.13 ± 0.19, 50.74 ± 6.27, 0.16 ± 0.11, 20.11 ± 3.78, 1.26 ± 0.54, 6.11 ± 0.60 in A. ruthenus, respectively. Significant differences were observed in Na⁺, K⁺ and Cl^? concentrations in the seminal plasma as well as in sperm velocity. The percentage of motile spermatozoa did not show any significant difference between the two species. Comparing the results of this study with published literature data on sturgeon spermatozoa reveals that morphological and ultrastructural parameters of spermatozoa together with some parameters of the seminal fluid and spermatozoa velocity can be used in comparative spermatology to better understand inter‐species differences. The observed biochemical and physiological differences should be also considered for the development of methods for controlled reproduction and for sperm cryopreservation techniques.     

Semen cryopreservation of yellow croaker <Emphasis Type="Italic">Larimichthys polyactis</Emphasis>

The effects of various extenders and cryoprotectants on movable spermatozoa ratio (MSR), spermatozoa velocity (SV) and duration of spermatozoa motility (DSM) of post-thawed spermatozoa were examined. The MSR, SV and DSM of post-thawed sperm in artificial seminal plasma (ASP) extender were higher than those in marine fish Ringer’s solution (MFRS) extender (P < 0.01) and was not significantly different from that of fresh sperm. No significant differences were observed in the motility parameters between fresh spermatozoa and frozen-thawed spermatozoa cryopreserved with ASP extender supplement 10% EG (ethylene glycol) cryoprotectant. Using the above method, yellow croaker semen was cryopreserved with extender ASP and 10% EG. As a result, at the spermatozoa/egg ratio of 100,000:1, the fertilization rate and hatching rate of the frozen-thawed spermatozoa cryopreserved for 1 week or 1 year in liquid nitrogen (45.7 ± 3.2% and 27.2 ± 5.0% or 37.5 ± 4.4% and 27.2 ± 5.0%) were similar to that of fresh spermatozoa (51.0 ± 3.1% and 36.7 ± 2.2%). There was a small alternation of shape in cryopreserved spermatozoa compared with fresh spermatozoa. In conclusion, the optimal conditions for yellow croaker semen cryopreservation are ASP extender supplement 10% EG cryoprotectant. This is the first report on semen cryopreservation of yellow croaker Larimichthys polyactis.     

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.     

Comparison of semen collection techniques in goats

Characteristics of goat semen collected with the artificial vagina (AV), electroejaculator (EE), and Bailey ejaculator (BE) were compared. Semen was collected three times by each method from four bucks for a total of 36 separate collections. The semen was evaluated for volume, sperm concentration, mass activity (0 to 4, no activity to rapid wave motion), sperm motility (%), pH, and acrosome morphology (% normal). The means (± SEM) of semen samples collected with the AV, EE, and BE were volume-0.4 (±0.1), 0.7 (± 0.2), 0.6 (± 0.1) ml; concentration-4.6 (± 0.5), 2.1 (± 0.3), 1.4 (± 0.2) 10⁹/ml; mass activity-4.0 (± 0), 3.3 (± 0.3), 2.8 (± 0.2); motility-81 (± 2), 78 (± 2), 76 (± 2) %; pH-6.2 (± 0.1), 7.0 ± (0.2), 7.0 ± (0.2); and normal acrosomes-96 (± 1), 92 (± 3), and 88 (± 3) %, respectively. Semen collected with the AV was lower in volume than that collected with the BE (P < 0.05). The sperm concentration in semen samples collected with the AV was greater than those collected with the EE and BE (P < 0.05). Mass activity was greater and pH was less in AV samples than in EE or BE samples (P < 0.05). There was no difference in sperm motility of semen samples collected by the AV, EE, or BE. The average percentage of normal acrosomes was greater for AV than for BE samples (P < 0.05). Use of the EE and especially the BE resulted in increased vocalization by the goats and excessive muscular contractions of the rear limbs. The BE, in its present form (11.2 volts), is not recommended for semen collection in goats.