Comparing the cryoprotective effects of glycerol and DMSO, with or without Equex, on Asian elephant (Elephas maximus) spermatozoa.

The dynamics of sperm DNA stability in Asian elephant (Elephas maximus) spermatozoa before and after cryopreservation

The main aim of this study was to compare the motility and functional integrity of bull spermatozoa after single and double freezing and thawing. The viability and morphological integrity of spermatozoa selected by PureSperm density gradient centrifugation after cryopreservation of bovine semen in two commercial extenders (Experiment 1) and the function of bull spermatozoa before and after a second freezing and thawing assisted by PureSperm selection (Experiment 2) were examined. On average, 35.8 +/- 12.1% of sperm loaded onto the PureSperm density gradient were recovered after centrifugation. In Experiment 1, post-thaw motility and acrosome integrity were higher for spermatozoa frozen in Tris-egg yolk extender than in AndroMed, whether the assessments were made immediately after thawing [80.4 +/- 12.7 vs 47.6 +/- 19.0% motile and 78.8 +/- 8.3 vs 50.1 +/- 19.5% normal apical ridge (NAR), p < 0.05] or after preparation on the gradient (83.3 +/- 8.6 vs 69.4 +/- 15.9% motile and 89.5 +/- 7.2 vs 69.1 +/- 11.4% NAR, p < 0.05). For semen frozen in Tris-egg yolk extender, selection on the PureSperm gradient did not influence total motility but significantly improved the proportion of acrosome-intact spermatozoa. After the gradient, both the total motility and percentage of normal acrosomes increased for spermatozoa frozen in AndroMed (Minitüb Tiefenbach, Germany). In Experiment 2, there was no difference in sperm motility after the first and second freeze-thawing (82.9 +/- 12.7 vs 68.8 +/- 18.7%). However, the proportion of acrosome-intact spermatozoa was significantly improved by selection through the PureSperm gradient, whether measured by phase contrast microscopy (78.9 +/- 9.7 vs 90.4 +/- 4.0% NAR, p < 0.05) or flow cytometry (53.4 +/- 11.7 vs 76.3 +/- 6.0% viable acrosome-intact spermatozoa, p < 0.001). The improvement in the percentage of spermatozoa with normal acrosomes was maintained after resuspension in the cooling extender and cooling to 4 degrees C (88.2 +/- 6.2) and after re-freezing and thawing (83.6 +/- 6.56% NAR). However, flow cytometric assessment of the sperm membranes revealed a decline in the percentage of viable spermatozoa with intact membranes after the second freezing and thawing compared with after gradient centrifugation (76.3 +/- 6.0% vs 46.6 +/- 6.6%, p < 0.001) to levels equivalent to those obtained after the first round of freeze-thawing (53.4 +/- 11.7% viable acrosome-intact spermatozoa). Sperm movement characteristics assessed by computer-assisted analysis were unaffected in the population selected on the PureSperm gradients but declined after cooling of the selected and extended spermatozoa to 4 degrees C. There was no further change in these kinematic measurements after the cooled spermatozoa had undergone the second round of freeze-thawing. These results demonstrate that bull semen can be frozen and thawed, followed by a second freeze-thawing cycle of a population of spermatozoa selected by PureSperm, with retained motility and functional integrity. This points to the possibility of using double frozen spermatozoa in bovine artificial insemination programmes and to the potential benefits of PureSperm density gradient centrifugation for the application of cryopreserved bull spermatozoa to other biotechnological procedures such as flow cytometric sex sorting followed by re-freezing and thawing.

Asian elephant spermatozoa are sensitive to chilling and do not respond well to cryopreservation. The objectives of the present study were to: (1) determine whether cholesterol content can be modified by preincubation of Asian elephant spermatozoa with cholesterol-loaded cyclodextrin (CLC); and (2) assess the effects of CLC concentration(s), temperature at time of glycerol addition (22°C vs 4°C) and dilution medium on post-thaw sperm survival. Spermatozoa incubated with ≥1.5 mg CLC exhibited increased (P < 0.05) cholesterol concentrations. Pretreatment of spermatozoa with 1.5 mg CLC resulted in improvements (P < 0.05) in all post-thaw parameters. Glycerol addition at 4°C also improved all post-thaw parameters compared with 22°C. Dilution of thawed spermatozoa in an egg yolk-based medium improved (P < 0.05) motility compared with Ham's F-10 culture medium. In summary, our findings indicate that modifying cholesterol content within the plasma membrane improves the cryosurvival of Asian elephant spermatozoa. The development of an improved cryopreservation method that includes modification of membrane cholesterol and the addition of glycerol at 4°C, as reported in the present study, is an important step towards utilisation of cryopreserved spermatozoa in captive management of this species.

Knowledge about the acrosomal status of Asian elephant (Elephas maximus) sperm is extremely limited. The objective of this study was to evaluate the viability and acrosomal status of Asian elephant sperm following induction by calcium ionophore and heparin using propidium iodide (PI) and fluorescein isothiocyanate conjugated peanut agglutinin (FITC-PNA). Semen samples were collected from elephant bulls by manual stimulation. Semen was diluted with extender, cooled to 4 degrees C and transported to a laboratory for the experiment. Sperm cells were incubated in modified Tyrode's medium containing either 1 mM calcium ionophore or 10 mg/ml heparin for 5 h at 39 degrees C. Sperm recovered at the onset (0 h), 1, 2, 3, 4 and 5 h of incubation were simultaneously assessed for the viability and acrosomal status using dual staining of FITC-PNA and PI. Results were confirmed by transmission electron microscopy. A progressive increase in the proportion of live-acrosome reacted sperm was observed within 3 h of incubation in both treatment groups which slightly decreased at 4 to 5 h of incubation. At 1 to 3 h of incubation, the percentage of live-acrosome reacted sperm induced by calcium ionophore was higher (P < 0.05) than those induced by heparin and the control. However, there were no statistical differences at 4 to 5 h of incubation. A progressive reduction of the percentage of motile sperm was observed in the control as well as both treatment groups. Sperm motility decreased sharply when they were incubated in calcium ionophore compared with incubation in heparin and control groups. These results indicate that the occurrence of live-acrosome reacted sperm in the Asian elephant was induced by calcium ionophore at a rate higher than that induced by heparin.

The effects of seminal plasma (SP), derived from autologous, homologous and heterologous species (stallion, boar and dog) on chilled Asian elephant sperm quality, were determined. Semen was collected from eight males and samples with ≥30% motile spermatozoa were used in the study. Semen was diluted with Tris-glucose-egg yolk extender, supplemented with different SP types and preserved at 4°C for 48hr. Experiment 1 (n=31), showed that the presence of SP (autologous) helped to preserve sperm quality in terms of sperm motility and acrosome integrity (p<.05). Homologous SP did not result in better sperm quality than autologous SP. Heterologous SP from stallion provided higher sperm motility and velocities compared to autologous SP (p<.05). Experiment 2 (n=14) determined the effect of different SP from four stallions. All stallion SP gave higher (p<.05) results for motile spermatozoa and sperm velocities than autologous SP. In conclusion, the presence of SP helps preserve Asian elephant sperm quality and stallion SP supports the motility of Asian elephant spermatozoa during cold storage.

Evaluation of post-thaw Asian elephant (Elephas maximus) spermatozoa using flow cytometry: the effects of extender and cryoprotectant

Artificial insemination (AI) is a potentially useful tool for breeding captive elephants because it facilitates efforts to minimise inbreeding. However, cooled storage of elephant semen markedly reduces fertility. This study compared the effects on semen-quality parameters, including sperm DNA fragmentation, of storing elephant semen at 4°C or 15°C in a commonly-used diluent (TEST) or a diluent developed to protect against sperm DNA damage (BullMax). Storing elephant semen for >24 h in either extender at either temperature resulted in decreases in sperm motility, viability, acrosome integrity and DNA integrity (P < 0.05); the decrease in motility was especially rapid. A subjective impression of circular sperm movement in TEST was confirmed by a higher curvilinear velocity and amplitude of lateral head displacement, but lower straight-line velocity and linearity than in BullMax. Initial percentages of spermatozoa with fragmented DNA (%SDF) did not differ between extenders or temperatures, but the rate of increase in %SDF during a 48-h incubation at 37°C was higher in TEST than in BullMax (P < 0.05). In conclusion, BullMax allows more linear movement and better preserves DNA stability of stored elephant spermatozoa than TEST. Sperm DNA stability during incubation at 37°C is a promising, discriminative parameter for selecting semen storage conditions of bulls for elephant AI.

Sperm sex-sorting in the Asian elephant ( Elephas maximus)

Intact plasma and acrosome membranes and functional mitochondria following cryopreservation are important attributes for the fertilizing ability of spermatozoa. In the present study, functional and ultrastructural changes of Asian elephant spermatozoa after cryopreservation either in TEST + glycerol or HEPT + dimethyl sulphoxide (DMSO) were evaluated by fluorescent techniques and electron microscopy. Sperm frozen in TEST + glycerol had higher proportion of sperm with intact plasma (49.1 +/- 9.2% vs. 30.9 +/- 3.9%) and acrosomal (53.7 +/- 4.9% vs. 35.8 +/- 6.1%) membranes, as well as active mitochondria (57.0 +/- 7.2% vs. 42.0 +/- 5.0%) than those cryopreserved in HEPT + DMSO. The results obtained from electron microscopy were similar to those obtained by fluorescence microscopy. The percentage of normal spermatozoa was higher when spermatozoa were frozen in TEST + glycerol than those frozen in HEPT + DMSO (31.8 +/- 5.6 vs. 28.5 +/- 6.4). The ultrastructural alterations revealed by transmission electron microscopy could be classified as (i) distension of plasma membrane, while the acrosome was swollen; (ii) disruption or loss of plasma membrane, while acrosome was swollen with distended outer acrosomal membrane; (iii) disruption or loss of plasma and outer acrosomal membrane with leakage of acrosome content; (iv) extensive vesiculation of plasma and outer acrosomal membrane and leakage of acrosome content; (v) a complete loss of both plasma membrane and outer acrosomal membrane; and (vi) swelling of mitochondria. These findings suggest that the freezing and thawing procedure caused structural damage to elephant spermatozoa, especially in the plasma membrane, acrosome and mitochondria. Fluorescence and electron microscopic evaluations are potentially a powerful tool in the analysis of elephant spermatozoa after freezing and thawing.

The biotechnological potential for manipulating offspring sex in the rhinoceros and the elephant

Sperm cryopreservation offers a long-term preservation of male genetic materials for future assisted reproductive technologies. However, dramatic changes in temperature during freezing and thawing injure sperm cells. While motility is essential for AI and membrane integrity is crucial for in vitro fertilization (IVF), sperm DNA integrity is a common index of fertilizing capability required for AI, IVF and intracytoplasmic sperm injection (ICSI). In endangered felids died unexpectedly, attempts have been made to recover as many DNA intact spermatozoa as possible from epididymis and testis to increase the opportunity to produce offspring in future. Although sperm from caudal epididymis has shown retained fertilizing capability after freezing and thawing (27.3% conception rate after unilateral intrauterine insemination), sperm recovery from the corpus epididymis has been suggested as an alternative to increase the amount of preserved genetic materials. To improve epididymal sperm quality, pre-treatment with single-layer centrifugation resulted in selection of sperm cells with intact DNA while post-thaw treatment with extracellular ATP incubation promoted the blastocyst rate. Cold storage of domestic cat testis for 7days at 4°C demonstrated <1% of sperm cells with fragmented DNA. Moreover, isolated testicular sperm cells, stored for 7days at 4°C, produced after ICSI poorer percentages of cleavage, morula and blastocyst than the fresh control. In wild felids, a death-to-necropsy time of 2hr after a jungle cat (Felis chaus) aged 10years died during anaesthesia plus another necropsy-to-sperm recovery time of 25hr has been reported to yield the post-thawed testicular sperm with 22.2% intact DNA. In summary, the chromatin structure of feline ejaculated and epididymal sperm seems to be tolerated to cold storage and cryopreservation; thus, fertilizing capability is well protected. In contrast, the cat testicular sperm DNA is generally damaged through the cryopreservation.

Cryopreservation of Iberian pig spermatozoa. Comparison of different freezing extenders based on post-thaw sperm quality

Improved cryopreservability of stallion sperm using a sorbitol-based freezing extender

Simple SummaryRooster sperm cryopreservation is the preferred technique for genetic preservation, particularly in indigenous breeds whose numbers are decreasing. However, cryopreserved semen is highly damaged, mainly from cold shock and oxidative stress during freezing, leading to membrane and sperm damage. It is necessary to protect sperm membranes against peroxidative damage to improve semen quality and fertility. This study aimed to determine the optimal ginseng concentration used as a freezing extender in cryopreserved Thai native rooster semen. Ginsenosides are active components extracted from ginseng that exert antioxidant effects that inhibit oxidative stress. Our results showed that ginseng extract dosage in the freezing extender influenced frozen-thawed semen quality, improved motility and membrane integrity, reduced oxidative stress caused by the freezing process, enhanced enzyme activity in seminal plasma, and increased fertility in rooster semen cryopreservation.To the best of our knowledge, this study is the first to determine the effect of ginseng as an antioxidant supplement in freezing extenders on the quality of cryopreserved rooster semen. Semen samples were collected from 40 Thai native roosters (Pradu Hang Dum) using the dorso-abdominal massage method and then pooled and divided into five groups according to the concentrations of ginseng supplementation (0, 0.25, 0.50, 0.75, and 1 mg/mL) in a freezing extender. The semen suspensions were loaded into a medium straw and cryopreserved using the liquid nitrogen vapor method. The post-thaw semen was evaluated for sperm quality (sperm motility and membrane integrity), seminal plasma characteristics (lipid peroxidation, superoxide dismutase [SOD], catalase [CAT], and glutathione peroxidase [GPx]), and fertility. The results showed that ginseng extract supplementation at 0.25 mg/mL yielded the highest total motility, progressive motility, and membrane integrity (59.47%, 30.82%, and 48.30%, respectively; p < 0.05) in cryopreserved rooster semen. Higher malondialdehyde concentrations were observed in the control group than in the other groups (p < 0.05). SOD, CAT, and GPx increased compared with those in the control group (p < 0.05). The results showed that the fertility rate with 0.25 mg/mL of ginseng was higher than that of the control group (62.80% vs. 46.28%: p < 0.05). In conclusion, supplementation with 0.25 mg/mL of ginseng is recommended as an alternative component to the freezing extender to improve rooster semen cryopreservation.

Evaluation of sperm acrosome reaction in the Asiatic elephant