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The long-term cryopreservation of the germplasm (spermatozoa and oocytes) of threatened species offers flexibility in their genetic management and security against disasters or outbreaks of disease that might seriously affect subpopulations. A wide range of infectious diseases can, however, be transmitted via artificial insemination, and the risk of infectious diseases being spread via manipulated germplasm needs to be carefully managed in order to avoid fertility problems in the dams and decreased survival of offspring. Furthermore, accidental introduction of exotic microorganisms into ecosystems remain a major threat. The aims of the present study were to assess the impact of diseases transmissible by sperm on the establishment of germplasm banks for the Iberian ibex, a wild mountain ungulate, and to determine the influence of the presence of these pathogens on sperm functionality. Blood and sperm samples were obtained from 52 mature ibex males legally shot in southern Spain. Sperm motility, morphological abnormalities, acrosome integrity and plasma membrane integrity were assessed for each sample to determine in vitro sperm quality. All serum samples underwent serological analysis for bovine herpes virus type I, bluetongue virus (BTV), bovine leukaemia virus, caprine arthritis-encephalitis virus, pestivirus, Brucella, Coxiella burnetii, Chlamydiophila abortus, Mycoplasma agalactiae and Borrelia burgdorferi. The highest prevalence (30.7%) was recorded for B. burgdorferi, followed by C. burnetii (13.4%). A total of 734 sperm doses containing approximately 200 × 10 spermatozoa each were frozen in straws. Forty-five straws (6.1% of the total number) came from animals seropositive for diseases listed in the Terrestrial Animal Code of the OIE for collection and processing of bovine and small ruminant semen (in this case, pestivirus and BTV). A total of 271 frozen straws (36.9% of the total of frozen straws) were provided by animals seropositive for pathogens potentially transmissible by semen not included in the above OIE code (in this case, B. burgdorferi and C. burnetii). The values of sperm variables were not affected by seropositivity for any of the pathogens transmissible by sperm. [ABSTRACT FROM AUTHOR]

Abstract: Insufficient cryoprotectant permeation is one of the major obstacles for successful fish embryo cryopreservation. The purpose of this study was to test the effectiveness of osmotic and chemical treatments to enhance cryoprotectant uptake by fish embryos. Japanese whiting Sillago japonica embryos at the somites and tail elongation stages were treated with hyperosmotic sugar solutions (1 M trehalose and sucrose) for 2–6 min, or a permeating agent (2–6 mg/mL pronase) for 30–120 min, and then impregnated with 10–15% DMSO in artificial sea water or aqueous solutions containing inorganic salts (0.125–0.25 M MgCl2 and CaCl2). The viability of the embryos after the treatments was estimated from hatching rates and the internal DMSO concentration was measured by HPLC. Treatment with trehalose for 3 min prior to impregnation with DMSO enhanced the uptake of the cryoprotectant by 45% without significantly affecting embryo viability, whereas pronase had no noticeable effect on cryoprotectant permeation. Incorporation of DMSO into the embryos was enhanced by 143–170% in the presence of 0.25 M MgCl2 and 0.125 M CaCl2 compared to sea water. A combination of treatments with trehalose and MgCl2 was even more effective in promoting DMSO permeation (191% compared to untreated embryos). Tail elongation embryos were less tolerant of the treatments, but had higher DMSO impregnation. In conclusion, the use of trehalose (as dehydrating agent) and MgCl2/CaCl2 (as a vehicle during impregnation) greatly promoted cryoprotectant uptake and may be a promising aid for the successful cryopreservation of fish embryos. [Copyright &y& Elsevier]

Abstract: The objectives of the present study were to test the effect of coconut water solution and TES–TRIS on the seminal coagulum liquefaction, sperm activation in fresh diluted semen, and on the cryopreservation of semen from capuchin monkeys (Cebus apella). Semen was collected from six males by electro-ejaculation, diluted in TES–TRIS or coconut water solution (CWS), and incubated at 35°C until the coagulated fraction of the semen was completely liquefied. In the experiment I, after liquefaction, samples were diluted in TES–TRIS or CWS, plus 6 and 10mM/mL of caffeine. Sperm motility and vigor were evaluated during 5h. For experiment II, after liquefaction, semen samples were extended in TES–TRIS (3.5% glycerol in the final solution) or CWS (2.5% glycerol in the final solution), cryopreserved and stored in liquid nitrogen for 1 week. The seminal coagulum was liquefied in (mean±SDM) 4.5±1.7 and 2.8±1.1h in TES–TRIS and CWS, respectively. Sperm were motile in TES–TRIS and CWS for 5.0±1.4 and 1.0±0.5h, respectively. The mean motility in this period was 38±22% (TES–TRIS) and 22.0±16.0 (CWS). Motility increased after caffeine addition only in samples diluted in CWS containing 6mM (22.5±16.0) or 10mM (28.0±19.0) caffeine. Post-thaw live sperm percentage was 26.2% in TES–TRIS and 13.2% in CWS. For cryopreservation of semen from C. apella TES–TRIS (3.5% glycerol) was more appropriate than CWS (2.5% glycerol). CWS+caffeine potentially increase sperm motility and may be useful in artificial insemination of fresh diluted semen. [ABSTRACT FROM AUTHOR]

Abstract: Artificial insemination (AI) with liquid-stored spermatozoa and sperm cryopreservation using directional freezing (DF) have been successful in the beluga. This study built on this foundation to develop a deep intra-uterine AI technique with frozen-thawed semen in beluga. Forty-two ejaculates from one male were cryopreserved using DF technology and subsequently used for 10 insemination attempts with seven females. Percentage pre- and post-thaw progressive motility and viability were (mean ± SD) 73.0 ± 12.2, 38.4 ± 8.8, 88.0 ± 0.1, and 59.3 ± 15.7%, respectively. A series of GnRH injections (3 x 250 μg, IV, 1.5 to 2 h apart) were used to induce ovulation, once a growing follicle >2.5 cm in diameter was visualized via trans-abdominal ultrasonography. Artificial insemination was performed at 30.1 ± 3.8 h post-initial GnRH injection with semen deposited in the uterine horn, 92.6 ± 16.2 cm beyond the genital opening using a flexible endoscope. The external cervical os (cEOS) was located beyond a series of 5 to 10 vaginal rings, 44.8 ± 9.3 cm from the external genital opening. The internal bifurcation of the uterus was 27 ± 6.8 cm beyond the cEOS. Ovulation occurred at 8.5 ± 7.6 h post-AI. Two of 10 inseminations (20%) resulted in pregnancy. The first pregnancy resulted in twins; both calves were born 442 d after AI, with one surviving. The second pregnancy is ongoing. These findings represent the first successful application of AI using frozen-thawed semen in beluga, and are important examples of how assisted reproductive technologies can provide tools for the global management of threatened species. [Copyright &y& Elsevier]

Abstract: Knowledge about the reproduction of the endangered pygmy hippopotamus is almost non-existent. This study takes the first step toward changing this by devising a protocol for the collection, evaluation, and short-term preservation of semen of this endangered species. Semen was collected successfully from seven bulls by electroejaculation, using a specially designed rectal probe. Mean ± SEM values of native sperm parameters from combined best fractions were: motility—80.0 ± 4.1%, concentration—2421 ± 1530 × 106 cells/mL, total collected cell number—759 ± 261 × 106 cells, intact acrosome—87.8 ± 1.2%, intact morphology—52.7 ± 4.3%, and, for some, hypoosmotic swelling test—79.3 ± 4.4% and seminal plasma osmolarity—297.5 ± 3.3 mOsm. Seven different extenders were tested for sperm storage under chilling conditions: Berliner Cryomedium (BC), Biladyl®, modification of Kenney modified Tyrode’s medium (KMT), MES medium, Androhep®, boar M III™ extender and Human Sperm Refrigeration Medium. While differences between males were apparent, the BC was consistently superior to all other extenders in sperm motility and facilitated storage for 7 d with up to 30% motility and some motility even after 3 weeks. With this knowledge in hand, the obvious two directions for future research are to conduct artificial insemination and to develop a technique for sperm cryopreservation. [ABSTRACT FROM AUTHOR]

Abstract: This study aimed to establish artificial insemination (AI) protocols to predictably initiate pregnancy during the breeding season in the European brown hare (EBH) (Lepus europaeus PALLAS, 1778). Semen was collected from seven captive and eight free-ranging males by means of electroejaculation. Semen from the free-ranging males was cryopreserved using directional freezing. Total motility/integrity of fresh and frozen-thawed semen was 91.6%/87.7% and 46.9%/53.8%, respectively. Ovulation was induced in ultrasonographically preselected females using a gonadotropin-releasing hormone analogue. Each female was inseminated with 1mL fresh (Group A, n=16) or frozen-thawed semen (Group B, n=9) at a concentration of 100×106 spermatozoa/mL. The use of ultrasonography (10 to 22MHz) confirmed the intracervical semen deposit, the success of artificial ovulation induction (formation of postovulatory corpus luteum), and permitted the monitoring of individual pregnancies. Although sperm motility/integrity was significantly different between groups, no significant difference was detected in conception rates (A, 87.50%; B, 77.78%). Because of embryonic resorption, there was a slight difference in fertility rate between groups (A, 62.5%; B, 77.78%). Overall, AI in captive EBH using fresh and frozen-thawed semen achieved successful fertility rates. Long-term cryopreserved semen was used to bring new genetic material from the wild into a genetically limited captive population without extensive animal transport. Therefore, AI has the potential to enhance breeding programs for EBH especially when cryopreserved semen from wild donors is used. [Copyright &y& Elsevier]

Abstract: Synchronization of estrous and fixed-time artificial insemination (FTAI) was conducted during the reproductive season of 2008 (March–April) in a local red deer breeding farm in Argentina. Multiparous suckling hinds (n =38) were artificially inseminated following hormonal treatment (intravaginal sponge containing 100mg of medroxiprogesterone acetate). At the time of sponge removal (day 12) 250 IU of eCG and 500μg of PGF2α were given to each hind. The FTAI was performed at 48–55h after device removal with cryopreserved semen imported from New Zealand. Rectal-transcervical AI method (similar to that in cattle) was performed and semen was deposited within the uterine body (n =28) or the cervix (n =10). Pregnancy was diagnosed by means of ultrasonography 44 days after FTAI. The overall pregnancy rate was 36.8% (14/38). Percentage of does that became pregnant with intrauterine seminal deposition was 42.9% (12/28) whereas pregnancy rate in the hinds with intracervical AI was 20% (2/10; P =0.27). [Copyright &y& Elsevier]

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Abstract: Fishes of the genus Xiphophorus are well-studied biomedical research models, and some species, such as X. couchianus, are currently listed as endangered in the wild. Sperm cryopreservation in these live-bearing fishes has begun recently. Thus far, live young have been produced with cryopreserved sperm only in one species (Xiphophorus helleri). In this study, the goal was to develop a practical protocol for sperm cryopreservation of Xiphophorus couchianus, and to produce live young with cryopreserved sperm. Sperm were collected by crushing of testis in Hanks'' balanced salt solution at an osmolality of 500 mOsmol/kg (HBSS500), and were cryopreserved with 14% glycerol (v/v) as cryoprotectant at a cooling rate of 20 °C/min from 5 to −80 °C in 250-µL French straws. For artificial insemination, samples were thawed at 40 °C for 5 s in a water bath, washed once using fresh HBSS500 by centrifuging at 1000 g for 5 min at 4 °C, concentrated into ~5 µL, and injected into virgin females of Xiphophorus maculatus. The inseminated females were monitored for 90 days for subsequent discharge of live young. Results from 2006 and 2007 showed considerable male-to-male variation in post-thaw motility (from 1 to 70%). Offspring were produced by cryopreserved sperm in two tanks (of three) at 36 and 66 days after insemination in 2007. Paternity was confirmed via phenotypes (body color) and genotypes (microsatellite genetic marker) of the hybrid offspring. Overall, a practical protocol for sperm cryopreservation and artificial insemination is provided to preserve X. couchianus, which is an important biomedical research model, and also currently listed as an endangered species in the International Union for Conservation of Nature (IUCN) red list. [Copyright &y& Elsevier]

Abstract: The first successful artificial insemination (AI) in a rhinoceros was reported in 2007 using fresh semen. Following that success, we decided to evaluate the possibility of using frozen-thawed semen for artificial insemination. Semen, collected from a 35–36 year old Southern white rhinoceros (Ceratotherium simum simum) in the UK was frozen using the directional freezing technique. This frozen semen was used in two intrauterine AI attempts on a 30 years old female rhinoceros in Hungary. The first attempt, conducted 30 days postpartum with an insemination dose of ∼135×106 motile cells, failed. The second attempt, conducted two estrus cycles later with an insemination dose of ∼500×106 motile cells, resulted in pregnancy and the birth of a healthy offspring. This represents the first successful AI using frozen-thawed semen in a rhinoceros, putting it among very few wildlife species in which AI with frozen-thawed semen resulted in a live birth. The incorporation of AI with frozen-thawed semen into the assisted reproduction toolbox opens the way to preserve and transport semen between distant individuals in captivity or between wild and captive populations, without the need to transport stressed or potentially disease carrying animals. In addition, cryopreserved spermatozoa, in combination with AI, are useful methods to extend the reproductive lifespan of individuals beyond their biological lifespan and an important tool for managing genetic diversity in these endangered mammals. [Copyright &y& Elsevier]

Abstract: Efforts toward the conservation and captive breeding of wildlife can be enhanced by sperm sorting and associated reproductive technologies such as sperm cryopreservation and artificial insemination (AI). Sex ratio management is of particular significance to species which naturally exist in female-dominated social groups. A bias of the sex ratio towards females of these species will greatly assist in maintaining socially cohesive groups and minimizing male–male aggression. Another application of this technology potentially exists for endangered species, as the preferential production of females can enable propagation of those species at a faster rate. The particular assisted reproductive technology (ART) used in conjunction with sperm sorting for the production of offspring is largely determined by the quality and quantity of spermatozoa following sorting and preservation processes. Regardless of the ART selected, breeding decisions involving sex-sorted spermatozoa should be made in conjunction with appropriate genetic management. Zoological-based research on reproductive physiology and assisted reproduction, including sperm sorting, is being conducted on numerous terrestrial and marine mammals. The wildlife species for which the technology has undergone the most advance is the bottlenose dolphin. AI using sex-sorted fresh or frozen-thawed spermatozoa has become a valuable tool for the genetic and reproductive management of captive bottlenose dolphins with six pre-sexed calves, all of the predetermined sex born to date. [Copyright &y& Elsevier]

Experimental insemination was performed using artificially produced low-motility sperm. A mathematical model was applied to the results of the insemination in order to clarify the relationship between sperm motility, the density of sperm and the fertilization rate of eggs. In the model, the probability of fertilization by individual spermatozoa was a function of sperm density in the insemination solution. The results showed that the probability of fertilization clearly decreased with increased sperm density, and the maximum possible fertilizing rate by increasing the sperm density was constrained by the proportion of motile sperm (% motility). The model was also applied to the results of insemination tests of cryopreserved sperm in order to evaluate the fertilizing capacity of cryopreserved sperm. It was proven that cryopreserved sperm needed a higher density to obtain the maximum fertilization rate compared with fresh sperm, and it was anticipated that the ratio of the motile inseminated cryopreserved sperm should be more than 5.0% to achieve an egg fertilization rate greater than 90%. [ABSTRACT FROM AUTHOR]

The endangered black-footed ferret (Mustela nigripes) has benefited from artificial insemination; however, improved sperm cryopreservation protocols are still needed. The present study focused on identifying factors influencing gamete survival during processing before cryopreservation, including: (1) the presence or absence of seminal plasma; (2) temperature (25C v. 37C); (3) type of medium (Ham's F10 medium v. TEST yolk buffer [TYB]); (4) cooling rate (slow, rapid and ultra-rapid); and (5) the presence or absence of glycerol. Seminal plasma did not compromise (P > 0.05) sperm motility or acrosomal integrity. Sperm motility traits were maintained longer (P 0.05) acrosomal integrity. Overall, TYB maintained optimal (P 0.05), indicating that 4% glycerol was non-toxic to ferret sperm; however, glycerol failed to ameliorate the detrimental effects of either rapid or ultra-rapid cooling. The results of the present study demonstrate that the damage observed to black-footed ferret spermatozoa is derived largely from the rate of cooling. [ABSTRACT FROM AUTHOR]

Abstract: Ten adult male spotted deer were monitored over a 2-year interval to determine seasonal variations in testicular size, semen characteristics and serum testosterone concentrations, and to determine if there was an association between season and type of antler. Mean (±S.E.M.) testicular volume (118.8±4.6cm3), serum testosterone concentration (1.2±0.1ng/mL), semen volume (4.1±0.6mL), sperm concentration (338.3±24.9×106 mL−1), percentage of morphologically normal sperm (79.1±2.8%), and percentage of motile sperm (66.5±1.5%) were higher (P <0.05) in hard antler deer (peaked from March to May) than in deer with velvet antlers or in deer in which the antler has been shed. Thus, March–May was considered the physiologic breeding season for these deer; at this time, all stags had hard antlers. Furthermore, a Tris-citrate-based semen extender containing 4% glycerol and 20% egg-yolk was adequate for cryopreservation of semen. Estrus was induced with an implant containing norgestomet, timed transcervical AI was done with fresh semen, and 3 of 10 females were pregnant at 60 days, with fawns born 120 (premature), 240 and 243 days after AI. These results were considered a model for the use of assisted reproductive techniques to conserve other critically endangered deer species of India. [Copyright &y& Elsevier]

To develop an appropriate cryopreservation protocol for spermatophores of black tiger shrimp, Penaeus monodon, three cryoprotectants (dimethyl sulphoxide (DMSO), methanol (MeOH) and ethylene glycol (EG)) at two concentrations (5% and 10%) were examined. Artificial implantation of spermatophores was also carried out to assess the fertilizing ability of fresh and post-thaw spermatophores. Spermatophores were collected during consecutive regenerations (15-day intervals) and assessed for qualitative and quantitative changes and also for fertilizing ability by implantation. The mean fertilization rate for artificial insemination using post-thaw spermatophore was 79.9±3.7%, lower than the fertilization rates observed for artificial implantation using fresh spermatophore and natural mating. Mean hatch rates for fresh spermatophore, frozen-thawed spermatophore and natural mating were 88.8±0.6%, 87.8±0.4% and 88.3±0.5%, respectively; and there was no difference among the three groups. The mean fertilization rate of spermatophores collected during the first stripping was higher (90.6±0.6) than during the second stripping (85.7±2.6), but the mean hatch rate was not different between the two strippings. The highest mean sperm viability (79.7±0.4%) was obtained from DMSO (5%), with no survival observed in the 10% MeOH treatment. Spermatophore weight, total sperm count and percentage of abnormal sperm were not different between spermatophores collected at the first and second stripping. This is the first study to report high fertilization and hatch rates from cryopreserved spermatophore using artificial implantation of spermatophore before spawning. [ABSTRACT FROM AUTHOR]

Artificial insemination with frozen-thawed buck semen shows variable results which depend on many factors related to semen quality and the cryopreservation processing. We conducted experiments based on a new freezing method, directional freezing, of large volumes (8 ml). In the first experiment semen from three Saanen bucks, ages 1–2-years-old and genetically selected for milk improvement, was frozen individually. Two to three-years-old Saanen females (n = 164) were synchronized with controlled internal drug release (CIDR), pregnant mare serum gonadotrophin (PMSG) and prostaglandin. Double cervical inseminations were performed with frozen-thawed semen and fresh semen as control. In the second experiment we used pooled, washed frozen semen to examine the effect of washed seminal plasma.The motility after washing was 80–90% and after thawing was 55–65% for all bucks. The sperm concentration increased with the collections and the advance into the breeding season from 1.9 × 109 to 4.4 × 109 cell/ml average. Two inseminations were carried out at 8 h intervals. The first insemination was performed at 32 h after CIDR withdrawal with fresh and frozen-thawed semen. Pregnancy rates were assessed by ultrasonography conducted 40 and 90 days post-insemination (from three bucks). Results were 58, 67, 50% with fresh semen, and for frozen semen were 33, 37 and 53%; these results were significantly different in one of the three bucks (P < 0.005). In the second experiment with pooled, washed semen the pregnancy rate was 41.6%, which compared with the average results of the frozen semen in the first experiment 38.9% no significant difference was found. We conclude that freezing buck semen in large volumes (8 ml) is possible. Cryobanking of buck semen will facilitate a genetic breeding program in goats and preservation of biodiversity. Washed semen did not improve the fertility of the semen when Andromed bull extender is used. [Copyright &y& Elsevier]

This paper reports on cryopreservation of sperm from turbot (Scophthalmus maximus). The effects of various extenders, cryoprotectants and sperm–egg insemination ratios on motility score and/or fertilization capacity of post-thaw turbot spermatozoa were examined to optimize cryopreservation procedures. Post-thaw motility of frozen sperm obtained with extender TS-2 was higher than those achieved with extenders D-15 and modified plaice Ringer solution (MPRS). The most effective cryoprotectant was determined to be 10% dimethyl sulfoxide (DMSO). Fertilization of small egg batches (2 ml eggs) with frozen sperm resulted in average fertilization rate (FR) (70.1±8.9%) and hatching rates (HR) (46.8±5.2%) similar to the fertilization rates (74.7±8.0%) and hatching rates (47.5±6.8%) of fresh sperm. The minimal density of frozen sperm required to obtain satisfactory fertilization rate was determined to be 2000:1 (sperm/egg). Fertilization of larger egg batches (40 ml eggs) with sperm frozen in 1.8-ml cryovials provided similar fertilization rates (71.6±7.3%) to that of the fresh sperm (76.2±5.7%), whereas the hatching rates (34.7±3.6%) of eggs fertilized with frozen sperm were slightly lower than those of fresh sperm (41.1±3.7%). Our results demonstrated that cryopreservation technique for turbot sperm in 1.8-ml cryovial could be used for hatchery purposes. [Copyright &y& Elsevier]

Reports that sperm cryopreservation and artificial insemination are important management tools for giant panda breeding and the preservation of extant genetic diversity. Examination on the influence of freeze-thawing on sperm function and capacitation; Assessment of sperms from a number of giant pandas before and after rapid cryopreservation; Discovery on the ability of giant panda spermatozoa to undergo capacitation with or without chemical accelerators and withstand a rapid cryopreservation protocol.

The ability to cryopreserve epididymal sperm from mature postmortem bucks has long been of interest to both wildlife conservationists and deer ranchers. At present, there is loss of valuable genetics from the hunter harvest of trophy males. Increasing adult body weight and antler size of adult males would be of substantial economic value to the deer hunting industry. In this preliminary trial, 6 yearling pen-raised Whitetail does (47 to 58 kg), in good body condition, were isolated from all bucks prior to the onset of the fall breeding season. Females were synchronized for AI with a 14-day caprine CIDR and 200 IU of eCG (IM) at the time of CIDR removal. Does were timed AI 60 to 63 h after eCG with one 0.5-mL straw of frozen–thawed Whitetail sperm. All sperm used for AI were harvested from a single mature Whitetail buck that was hunter harvested during the previous hunting season. Within 3 h after death, the testes with scrotum were removed, enclosed in a plastic Ziploc bag, and then placed in a Styrofoam ice chest containing frozen cold packs. The ice chest was transported to the laboratory where sperm were extracted at 4C in the late evening (<12 h postmortem) by flushing the cauda epididymides with Triladyl one-step extender (Minitube, Verona, WI, USA) in a retrograde flow from a small incision made in the cauda. The sperm–Triladyl mixture was flushed from the cauda incision into a sterile 50-mL tube using a 10-mL plastic syringe modified by heating and then stretching the tip until small enough to thread into the vas deferens. The sperm plus extender was then held at 4 to 10C for 12 h and frozen at a concentration of <50 million/straw using a commercial bull freezing protocol (Genex Custom Collection Center, Baton Rouge, LA, USA). A random sample of straws was then thawed, resulting in an overall post-thaw motility of 60%. The remaining straws were left frozen in liquid nitrogen until the next breeding season. On the first of December, does (n = 6) were given 0.1 mL of Domosedan (Pfizer Animal Health, Groton, CT, USA) IV and inseminated transcervically using a modified caprine speculum. All does were handled in a custom-built deer barn, and AI was performed by one technician in a drop-bottom deer chute (Deer Handler; Delclayna, Alberta, Canada). At 48 days after AI, 3 of the 6 does (50%) were diagnosed pregnant by transrectal ultrasonography. All pregnant females gave birth, producing 5 offspring (1 male and 1 female singletons and a set of mixed sex triplets) that ranged from 1.9 to 4.3 kg and had a mean gestation length of 196 days (range = 190–203 days). In summary, results indicate that live offspring can be produced from epididymal sperm harvested from mature hunter-harvested Whitetail bucks. Further experiments are needed to optimize techniques and protocols for the harvesting and usage of these gametes. [ABSTRACT FROM AUTHOR]