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Abstract: Bottlenose dolphins (Tursiops truncatus) undergoing natural breeding and artificial insemination (AI) were examined to characterize serum progesterone concentrations and determine relationships among age, parity, and reproductive outcome. Progesterone profiles of five cycle types (n = 119 total cycles from 54 animals) were characterized as follows: (i) conception and production of a live term calf (conceptive-term, n = 73); (ii) conception and abortion after Day 60 (conceptive-abortion, n = 12); (iii) unknown conception status with prolonged, elevated progesterone and absence of a fetus (conceptive-unknown, n = 14); (iv) conception failure with normal luteal phase progesterone concentrations (non-conceptive, n = 14, AI cycles only); and (v) conception failure with progesterone insufficiency occuring after spontaneous ovulation or owing to premature ovulation induction using GnRH (non-conceptive-PI, n = 6, AI cycles only). By Day 21 post-insemination (PI), progesterone concentrations were similar (P > 0.05) among conceptive-term, conceptive-abortion and conceptive-unknown, and higher (P < 0.05) for conceptive-term than non-conceptive and non-conceptive-PI cycles. Progesterone concentrations of known conceptive cycles peaked by Week 7 PI (P < 0.05) and remained elevated for the remainder of pregnancy (Weeks 8 up to 54, ≥5 days pre-partum). During midpregnancy (Days 121–240), conceptive-term cycles had higher (P > 0.05) progesterone concentrations than conceptive-abortion and unknown conception status cycles. Parity was not associated with reproductive outcome based on cycle type (P > 0.05). Age of females in conceptive-unknown (26.5 ± 10.1 yrs) and conceptive-abortion (22.1 ± 9.4 yrs) groups was higher (P < 0.05) than in conceptive-term (15.7 ± 7.2 yrs). The conceptive-unknown cycle type possibly represents undetected early embryonic loss occurring before Day 60 PI. Length of gestation using known conception dates was 376.1 ± 11.0 days and the range of this parameter (355–395 days) has implications for peri-parturient management procedures for the species. [Copyright &y& Elsevier]

Abstract: Tetraploidy is induced in rainbow trout by applying a pressure shock at a specific time point between insemination and first cleavage, or the first cleavage interval (FCI). Previous studies suggested that variation in the FCI among individuals and populations of fish prevents the identification of a single time point that can be used for all trout. In this study we confirmed the optimal time to apply pressure is 65±5% of the FCI. In addition, we found that variation in FCI of fish from a common environment can be within limits that allow a single time point to be established for that group of fish, if ova post ovulatory aging is taken into account. Aging of ova, either in vivo or in vitro, increased FCI to a degree that is a concern for tetraploid induction. The FCI was about 12min longer at 7days post ovulation, and 30min at 10–14days, than at 1day. The FCI for a group of fish was consistent throughout the spawning season. Survival to hatching and frequency of spinal abnormalities were similar for progeny of first and second generation tetraploid males, but survival was doubled and abnormalities reduced by approximately 90% in second generation tetraploid females compared with first generation females. All progeny of tetraploid by tetraploid crosses were determined to be tetraploids based on flow cytometry of embryonic cells. In summary, attention to ova aging and use of second generation female tetraploids allows efficient production of a tetraploid rainbow trout broodstock. [Copyright &y& Elsevier]

Abstract: Discriminating the male influence on egg fertilization in Penaeus monodon is needed so that accurate measures of male fertility can be developed and subsequently used to evaluate methods aimed at improving fertility. In this study, we employed a novel, pairwise comparison approach to discriminating the fertilization influence of males: female broodstock were artificially inseminated with one spermatophore each from a pair of males. We achieved 22 successful ‘spawnings’ involving selected pairs from 33 males, and estimated the proportion of embryos fertilized by each paired male by individual genotyping of embryos. The non-inseminated, ‘twin’ spermatophore was used to estimate measures of male fertility; sperm number and the number of normal sperm of each inseminated spermatophore. Relationships between measures of male fertility and egg fertilization were then evaluated. Using this approach we were able to show that the egg fertilization potential of males could not be determined simply by total sperm number or the number of normal sperm (as determined by gross morphology under light microscopy). We also found extremely high variability in fertilization between paired males. The results highlighted male infertility issues (e.g. sperm viability) that are not simply overcome by greater sperm numbers. Our novel approach provides a method that can be used to determine whether more complex measures of male fertility can reliably predict egg fertilization potential. [Copyright &y& Elsevier]

Abstract: Semen cryopreservation is an important technique for the banking of animal germplasm from endangered species and exploitation of genetically superior sires through artificial insemination. Being a member of bovidae family, bison semen has poor freezing ability as compared to dairy and beef bulls’ semen. This study was designed to quantify the damage to bison sperm at different stages of cryopreservation, and to determine the effects of extender (commercial Triladyl® vs. custom made tris-citric acid [TCA]) and freeze rate (−10, −25 and −40°C/min) on post-thaw quality of bison semen. Semen was collected from five bison bulls (three woods and two plains) via electroejaculation. In Experiment 1, semen was diluted in Triladyl® extender and frozen with freeze rate −10°C/min. Sperm motility characteristics were recorded in fresh, diluted, cooled (4°C) and freeze-thawed semen using computer-assisted sperm analyzer (CASA). In Experiment 2, semen was diluted in Triladyl® or TCA extender, and frozen with three different freeze rates, i.e. −10, −25 or −40°C/min. Thawing was performed at 37°C for 60s. Post-thaw sperm motility characteristics were assessed using CASA, and sperm structural characteristics (plasma membrane, mitochondrial membrane potential and acrosomes) were evaluated using flow cytometer, at 0 and 3h while incubating semen at 37°C. In Experiment 1, total and progressive motilities did not differ among pre-freeze stages of cryopreservation (P >0.05). However, sperm total and progressive motilities declined (P <0.001) in freeze-thawed semen by 35% and 42%, respectively, compared to after cooling (pre-freeze) semen. In Experiment 2, Triladyl®, as compared to TCA, yielded greater (P <0.05) post-thaw sperm total motility (41% compared to 36%) and progressive motility (34% compared to 29%) at 0h, respectively. The percent change in post-thaw sperm total and progressive motilities, VAP, VCL, VSL, IPM-high ΔΨm and IPM-IACR during 3h incubation at 37°C, was less (P <0.05) in TCA than in Triladyl®. There was an effect of freeze rate on post-thaw sperm average path velocity at 0h, and total motility, progressive motility, VCL, IPM and IPM-IACR at 3h were the greatest (P <0.05) when bison semen was frozen at −40°C/min. Likewise, the percent change in post-thaw sperm total and progressive motilities, during 3h incubation at 37°C, was less (P <0.05) in bison semen frozen at −40°C/min. All post-thaw bison sperm characteristics decreased (P <0.05) from 0h to 3h, during incubation at 37°C. In conclusion, the maximum damage to bison sperm occurred during freeze–thaw processes. Post-thaw total and progressive motilities of bison sperm were greater in Triladyl® at 0h whereas sperm survival was greater in TCA extender during 3h post-thaw incubation. Bison sperm had greater survival (P <0.05) when frozen at −40°C/min freeze rate. [Copyright &y& Elsevier]

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: A simple technique is described for isolating DNA from individual embryos or nauplii of the Black Tiger shrimp, Penaeus monodon. The protocol used a commercial prepGEM™ insect DNA extraction kit in combination with water bath ultrasonic disruption. DNA quality and yields were sufficient for multiplex PCR discrimination of up to six DNA microsatellites in a single test. The utility of the method was demonstrated in defining the parentage of 1279 embryos (7h old) spawned from 21 females and 684 newly-hatched N1 to N2 nauplii spawned from 12 females each inseminated artificially with the spermatophores of two different males. Overall, parentage assignments based on the six microsatellite markers were deduced to be 89.5±6.7% and 93.0±4.4% effective using DNA isolated from individual embryos and nauplii, respectively. The technique is simple, cost-effective and should provide a robust means of isolating DNA from individual embryos and nauplii of aquatic invertebrates suitable for PCR-based genotyping. [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: The feasibility of cross-breeding of Pacific white shrimp, P. (Litopenaeus) vannamei (PV) and blue shrimp, P. (Litopenaeus) stylirostris (PS), by means of artificial insemination, was evaluated. Intra-specific (pure species) crosses were used as control treatments: 1) ♂PV×♀PV and 2) ♂PS×♀PS. Reciprocal, inter-specific (hybrid) crosses 3) ♂PV×♀PS and 4) ♂PS×♀PV, were performed employing a variety of artificial insemination techniques. Twelve intra-specific inseminations of white shrimp (♂PV×♀PV) were performed. All inseminated females spawned eggs, ranging from 70,111 to 139,940 eggs per spawn. Five out of the 12 spawns produced nauplii (14,241 nauplii per spawn), with a mean hatch rate of 11.7%. For intra-specific blue shrimp crosses (♂PS×♀PS), 13 out of 14 inseminated females spawned eggs ranging from 112,349 to 282,141 eggs per spawn, and obtaining three fertile spawns with a mean hatch rate of 12.8% and 24,588 nauplii per spawn. For the inter-specific crosses, a total of 49 hybridization attempts were made, 27 for the cross ♂PV×♀PS and 22 for the reciprocal cross ♂PS×♀PV. In spite of trying artificial insemination in various ways, none of the hybridization attempts was successful; indicating that cross-breeding of white and blue shrimp is not possible by means of traditional artificial insemination techniques. [Copyright &y& Elsevier]

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]

Abstract: Our aim was to compare traditional breeding system, i.e. artificial insemination with singly-housing, to alternative breeding systems in farmed blue fox (Alopex lagopus or Vulpes lagopus) vixens. At the age of 7 weeks (i.e. at weaning), 48 randomly selected female blue fox cubs were divided into four experimental groups: (1) artificially inseminated singly-housed vixens in cages (AI-SC), (2) artificially inseminated pair-housed vixens in double-cages (AI-PC), (3) naturally bred pair-housed vixens with a male in triple-cages (NB-PC), and (4) naturally bred pair-housed vixens with a male in outdoor enclosures (NB-PE). The cubs were counted on days 1, 2, 3, 7, 14 and 49 postpartum and the reproductive performance per breeding (RPB) and per mated vixens (RPM) with its subcomponents were formed from these data. RPB was zero in both NB groups. In AI-PC and AI-SC, RPB was 1.3±2.5 and 4.1±4.7 cubs, respectively. RPB and the percentage of vixens that weaned cubs were lower in NB-PC and NB-PE than in AI-PC. In AI groups, both RPB and RPM at weaning tended to be better in singly-housed than in pair-housed blue fox vixens. No statistically significant differences were found between AI groups in the percentage of vixens without oestrus, barren vixens, vixens that lost all cubs or weaned at least one cub. Only in one pair (AI-PC) both vixens displayed communal breeding and weaned cubs. The present results show that vixens in traditional breeding system had better RPM than in any of the studied pair-housing systems. The current blue fox population has been effectively selected for cage-breeding and artificial reproduction, and therefore blue foxes generally reproduce well with artificial insemination after careful detection of oestrus in traditional breeding conditions. [Copyright &y& Elsevier]

Abstract: In captive Asian elephants, there is a strong need for production of female offspring to enhance reproduction, counter premature aging processes in female animals and reduce challenging management situations derived from husbandry of several bulls in one institution. Artificial insemination of flow cytometrically sex-sorted spermatozoa offers the possibility to predetermine the sex of offspring with high accuracy. The aims of this study were to determine a suitable semen extender and basic parameters for flow cytometrical sex-sorting of Asian elephant spermatozoa. In total 18 semen samples were collected by manual rectal stimulation from one bull. Sperm quality parameters and sex sortability of spermatozoa were evaluated after dilution in three semen extenders (MES-HEPES-skim milk, MES-HEPES, TRIS–citric acid) and DNA staining. MES-HEPES-skim milk was the only semen extender found suitable to sex Asian elephant spermatozoa. From 18 ejaculates collected, 12 were successfully sorted with a purity of 94.5±0.7% at an average sort rate of 1945.5±187.5 spermatozoa per second. Sperm integrity, progressive and total motility were 42.6±3.9%, 48.1±3.3%, 59.4±3.8% after DNA labelling, and 64.8±3.2%, 58.0±5.0%, 70.8±4.4% after sorting, respectively. After liquid storage of sorted spermatozoa for 12h at 4°C, sperm integrity, progressive and total motility were 46.4±5.2%, 32.2±4.2% and 58.2±3.9%, respectively. The obtained results provide a promising base to inseminate Asian elephants with sexed semen. [Copyright &y& Elsevier]

Abstract: The aim of the study was to show whether it is possible to induce ovulation in the hare by GnRH analogue administration and to carry out an effective artificial insemination (AI). The research was carried out during the breeding and non-breeding season. During the breeding season, plasma progesterone concentrations increased on the 4th day after intramuscular injection of GnRH analogue (buserelin), indicating induced ovulation and corpus luteum development. Prostaglandin F2α (dinoprost) was an effective luteolytic agent on day 9. During the non-breeding season, the GnRH analogue injection does not cause an increase of progesterone. The 17β-estradiol concentrations during the breeding and non-breeding season were similar. It was shown that after GnRH analogue administration and artificial insemination with semen diluted in Tris buffer extender 80% females delivered live young (39–43 days after artificial insemination), which proves the effectiveness of inducing ovulation in the hare by means of hormonal stimulation. [Copyright &y& Elsevier]

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: Short-term storage and cryopreservation of sperm are two common procedures in aquaculture, used for routine practices in artificial insemination reproduction and gene banking, respectively. Nevertheless, both procedures cause injuries affecting sperm motility, viability, cell structure and DNA stability, which diminish reproductive success. DNA modification is considered extremely important, especially when sperm storage is carried out with gene banking purposes. DNA damage caused by sperm storage is not well characterized and previous studies have reported simple and double strand breaks that have been attributed to oxidative events promoted by the generation of free radicals during storage. The objective of this study was to reveal DNA fragmentation and to explore the presence of oxidized bases that could be produced by oxidative events during short-term storage and cryopreservation in sex-reversed rainbow trout (Oncorhynchus mykiss) spermatozoa. Sperm from six males was analyzed separately. Different aliquots of the samples were stored 2h (fresh) or 5 days at 4°C or were cryopreserved. Then spermatozoa were analyzed using the Comet assay, as well as combining this method with digestion with two endonucleases from Escherichia coli (Endonuclease III, that cut in oxidized cytosines, and FPG, cutting in oxidized guanosines). Both storage procedures yielded DNA fragmentation, but only short-term storage oxidative events were clearly detected, showing that oxidative processes affect guanosines rather than cytosines. Cryopreservation increases DNA fragmentation but the presence of oxidized bases was not noticed, suggesting that mechanisms other than oxidative stress could be involved in DNA fragmentation promoted by freezing. [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: Approximately 503 of the known species of birds are classified as ‘endangered’ or ‘critical’. Captive propagation programs have proven useful in maintaining genetic diversity and restoring wild populations of certain species, including the Peregrine falcon, California condor and Whooping crane. Artificial insemination (AI) has the potential of solving problems inherent to reproductive management of small, closed populations of endangered birds, including dealing with demographic instability, physical and behavioral disabilities, sexual incompatibility, lack of synchrony, and need to maintain gene diversity. In this review, we address the necessary methods and factors that allow AI to be applied effectively to manage rare bird populations. It is clear that semen availability and quality are the greatest limiting factors to implementing consistently successful AI for birds. Behavioral sensitivity to animal handling and the ability to minimize stress in individual birds also are keys to success. Multiple, deep vaginal inseminations can improve fertility, particularly when semen quality is marginal. Laparoscopic methods of semen transfer also have produced fertile eggs. All of these practices leading to successful AI remain dependent on having adequate basic knowledge on female reproductive status, copulatory behavior, endocrine profiles and duration of fertility, especially as related to oviposition. The overall greatest challenge and highest priority is defining these normative traits, which are highly species-specific. [Copyright &y& Elsevier]

Abstract: Artificial insemination technology has revolutionized the domestic cattle breeding industry and allowed for the dissemination of valuable genetics worldwide. This technology has been adapted for use in many other taxa for the conservation of threatened and endangered species, but its use for the genetic management of small populations of deer, antelope and other non-domestic bovids has met numerous challenges and limited success. In practice, adaptation of domestic bovine AI protocols to other artiodactylids for genetic management has been limited by: (1) a lack of understanding of species-specific reproductive characteristics; (2) the inability to minimize handling stress; (3) pregnancy losses; and (4) regulatory challenges in semen importation. To date, AI protocols have been developed for seven species of cervid and 14 species of non-domestic bovids; recent developments in this technology has allowed greater use of AI for dissemination of genetics in farmed deer species. However, despite decades of research in the use of assisted reproduction for the conservation of antelope and other non-domestic bovids, even this simplest technique has not been used repeatedly for genetic management. [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]

Abstract: Finland blue fox (Alopex lagopus) has great reputation in pelt industry around the world for its large size and top-ranking fur quality; however, both the herd size and the average survival rate of purebred offspring are rather low in production systems in China. Surgical transfer of blue fox embryos was investigated as a means to increase the population fox and also as a possible means to conserve endangered canine species. The animals were chosen on the basis of synchrony in natural oestrus. During the reproductive season of blue fox, 59 embryos were flushed from 6 farmed donors 9–11 days after the first insemination, and 53 embryos were transferred surgically into the uteri of the 6 paired recipients with natural synchronized oestrous. Two of the recipients littered 46–49 days after embryo transfer; one gave birth to 7 pups and the other 1 pup. This report describes the first successful embryo transfer in the farmed blue fox in China. [Copyright &y& Elsevier]

Abstract: This study details the seminal traits in the rare Asiatic black bear, revealing that this species generally produces high quality, concentrated ejaculates containing sperm of high motility and morphological integrity and similar to other members of the Ursine lineage. Semen was collected by electroejaculation and 23 trials were performed on 18 bears. The mean values were obtained for volume (0.61ml), pH (7.1), sperm concentration (1049×106 ml−1), total sperm counts (502.8×106), motility percentage (63.8%), forward progressive status (3.5), abnormal sperm percentage (37.9%) and intact acrosome percentage (76.6%). [Copyright &y& Elsevier]

Abstract: The objective of this study was to develop AI and to achieve first time pregnancy in a nulliparous rhinoceros. For this, one 24-year-old irregular cycling female white rhinoceros was selected, which had never been mated. The endocrine function was monitored by faecal and serum pregnane analysis. Ultrasound determined the optimal day for AI by measuring follicle sizes of 2.0, 2.6, 3.0, 3.2cm on days −6, −4, −1, 0 of the induced oestrous cycle, respectively. AI was performed and ovulation induced when a pre-ovulatory-sized follicle was present using GnRH analogue, deslorelin. Fresh semen was deposited in the uterine horn using a patented AI catheter overcoming the hymeneal membrane and torturous cervical folds non-surgically. Moreover, ultrasound monitoring of the uterine involution and ovarian activity on days 16, 26, 30 postpartum facilitated the induction of and the AI on the first postpartum oestrous in a rhinoceros using GnRH analogue. Two consecutive pregnancies were achieved by AI for the first time in the rhinoceros. Pregnancies were diagnosed by elevated serum and faecal 20-oxo-pregnane concentrations. In addition ultrasound measured biometric parameters of the two foetuses on days 86 and 133 of gestation. Two female calves were born after 490 and 502 days of gestation, yet one calf was stillborn. AI in rhinoceros might now be used as assisted reproduction technology tool to boost critically small captive rhinoceros populations. [Copyright &y& Elsevier]

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]

A very large majority of farm animals express seasonal variations in their production traits, thus inducing seasonal availability of fresh derived animal products (meat, milk, cheese and eggs). This pattern is in part the consequence of the farmer's objective to market his products in the most economically favourable period. It may also be imposed by the season-dependent access to feed resources, as in ruminants, or by the specific requirements derived from adaptation to environmental conditions such as water temperature in fish. But seasonal variations in animal products are also the consequence of constraints resulting from the occurrence of a more or less marked seasonal reproductive season in most farm animal species including fish, poultry and mammals. Like their wild counterparts, at mid and high latitudes, most farm animals normally give birth at the end of winter-early spring, the most favourable period for the progeny to survive and thus promote the next generation. As a consequence, most species show seasonal variations in their ovulation frequency (mammals and fish: presence or absence of ovulation; birds: variations or suppression of laying rates), spermatogenic activity (from moderate to complete absence of sperm production), gamete quality (variations in fertilisation rates and embryo survival), and also sexual behaviour. Among species of interest for animal production, fishes and birds are generally considered as more directly sensitive to external factors (mainly temperature in fish, photoperiod in birds). In all species, it is therefore advisable that artificial photoperiodic treatments consisting of extra-light during natural short days (in chickens, turkeys, guinea fowl, sheep and goats) or melatonin during long days (in goats, sheep) be extensively used to either adjust the breeding season to animal producer needs and/or to completely overcome seasonal variations of sperm production in artificial insemination centres (mammals) and breeder flock operations (poultry, fish farming). Pure light treatments (without melatonin), especially when applied in open barns, could be considered as non invasive ones which fully respect animal welfare. [ABSTRACT FROM AUTHOR]

Abstract: Artificial insemination (AI) was performed in spider monkeys; these primates are vulnerable to extinction and usually do not reproduce spontaneously in captivity. Uterine cycles were followed by daily assessment of vaginal cytology, and corroborated a posteriori by concentrations of 17-β estradiol and progesterone, measured by radioimmunoassay (RIA), in fecal samples collected once daily. Five females between 13 to 27 years old were inseminated intravaginally (with fresh semen) twice each during the periovulatory phase (Days 9–12 of the menstrual cycle; Day 0, first day of menstrual bleeding), from September to the first 3 weeks of November (most fertile months). Transcervical AI was not useful in this primate because the liquid portion of the semen completely solidified instead of liquefying as in other primates. Pregnancies were apparently achieved in 5 of 14 attempts. One female became pregnant after the first round of inseminations, delivered a healthy infant, was inseminated and got pregnant again (subsequently aborted). One female aborted, apparently due to an intramural uterine leiomyoma. Another two females stopped menstruating for a few months, then restarted menstruating (these females may have been pregnant and aborted). In conclusion, in spider monkeys: (1) captivity-induced stress did not inhibit reproduction; (2) fecal steroid hormones were useful to assess cyclicity; (3) the semen coagulum, which apparently is a tightly packed and large reservoir of spermatozoa, must not be discarded but used in AI; (4) old female spider monkeys did not have cessation of reproductive function. [Copyright &y& Elsevier]

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]

The objectives were to determine the efficacy of the HeatWatch™ (HW) electronic estrus detection system for monitoring behavioral estrus (including duration and intensity) in red deer hinds and to evaluate pregnancy rate to AI after detected estrus. Red deer hinds (Cervus elephus; n=50) were allocated into two treatment groups: AI following synchronization (CIDR/PMSG) and observed estrus (induced estrus group: IE; n=25) or AI following the detection of natural estrus (NE; n=25) without hormonal treatment. Hinds were fitted with two HeatWatch (HW) electronic estrus detection transmitters, one above the tail (bottom) and one between the tuber coxae of the pelvic girdle (top), and visual observations for mounting activity began with the aid of young sterile red deer stags (18 months old) fitted with marking harnesses. Hinds in both groups were inseminated (10–12 h after observed estrus) with frozen–thawed red deer semen using a transvaginal/cervical AI approach. Following a 26-day period of AI, hinds were placed with a mature fertile stag for an additional 30-day natural breeding period. Pregnancy diagnosis was performed 57 and 86 days after the start of AI. While the hinds were housed with the young stags, 82% were detected in estrus by visual appraisal of stag crayon marks, but only 32% of these were detected by HW. In contrast, in the hinds housed with the mature stag, 93% detected in estrus by crayon marks were also detected by HW. The top HW transmitter consistently recorded more mounts (P<0.05) than the bottom transmitter. The pregnancy rate was numerically better in IE versus NE hinds (42% versus 29%, P>0.10). In summary, there were no differences (P>0.10) in the intensity (number) or duration of mounts (detected by HW) during estrus in IE versus NE hinds, and HW was most effective in detecting estrus in the presence of a heavier, mature stag versus a younger stag. When used in combination with transvaginal AI, an overall first-service pregnancy rate of 36.6% was achieved with AI of frozen–thawed semen in red deer hinds following detected estrus. [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]

The present work optimizes procedures for artificial insemination of European catfish, Silurus glanis L., with goals to increase collection of sperm and hatching rate. We address technical aspects of immobilizing solution (IS), activation solution (AS), process of insemination, activation of gametes and elimination of eggs stickiness with a practical solution for artificial propagation in hatcheries. Spermiation was stimulated and could be sustained over a period of 1 month after weekly carp pituitary (CP) injection of 5 mg kg-1 b.w. Males produced significantly the largest quantity of sperm, 0.12–0.13·109 spermatozoa kg-1 b.w., after third and fourth injections. The best IS for sperm was determined as solution containing 170–200 mM NaCl, 30 mM Tris–HCl, pH 7 and activation solution for activation of gametes containing 17 mM NaCl, 5 mM Tris–HCl, pH 7. The ratio of 1:2 rather than ratio of 1:1 in the control batch (volume of activation solution in milliliters/grams of eggs) increased the hatchability up to 68%. The highest hatching success of 82–88% from four individual females were found for sperm number of 800–80,000 spermatozoa egg-1, in comparison to 26% hatching at 80 spermatozoa egg-1. The best time for application of alcalase enzyme for elimination of egg stickiness was 3 min after gamete activation at concentration of 20 ml enzyme l-1 with exposure of 2 min. [Copyright &y& Elsevier]

Viable F1 hybrids were obtained from crosses of female Macrobrachium nipponense and male Macrobrachium hainanense involving spermatophore transfer and artificial insemination. This represents the first successful known case of hybridization of two Macrobrachium species by means of artificial insemination. The hatching rate was over 90%. About 20–60% of newly hatched larvae metamorphosed to postlarvae. The morphological characteristics of the hybrids resembled a combination of features of both parents. Malate dehydrogenase (MDH) and esterase (EST) isozyme electrophoresis indicated parents and F1 hybrids showed co-dominant expression of the paternal and maternal alleles controlling the isozymes and confirmed the hybridization. [Copyright &y& Elsevier]

We developed a simple, easy method with a microplate to artificially incubate Japanese crayfish (Cambaroides japonicus) eggs for their cultivation. We prepared 6-, 12-, 24-, 48-, and 96-well microplates containing sterile water heated to 5, 10, 15, and 20 °C. Fourteen experimental groups for each water temperature were prepared for each of different water volumes (0.125–10 ml) in each well. One embryonic egg was placed in each well. Experiments were also conducted with water collected from the lake where the eggs were harvested from and held at 15 °C. In the microplates with sterile water, high proportions of eggs hatched (60–100%) at 15 °C in all volumes of water, although the proportions of hatching were low (0–20%) at 5, 10, and 20 °C. All eggs died in the experiments that used lake water. We conclude that the 96-well would be the most effective size to hatch crayfish eggs in, because of its convenience. This method using a microplate is simpler and easier compared with methods of previous studies to artificially incubate crayfish eggs, and therefore it might be useful to incubate eggs of other freshwater crayfish species. [Copyright &y& Elsevier]

The application of artificial insemination techniques as a way to overcome the lack of mating of the closed thelycum Farfantepenaeus paulensis under large scale conditions is reported. The performance of artificially inseminated females (49.4±11.6 g) maintained in conventional versus unisex maturation systems was compared. Groups of unilaterally eyestalk-ablated females were artificially inseminated with one compound spermatophore soon after molting. The percentage of fertilized spawns increased from 26% before the use of artificial insemination to 57% afterwards. The reproductive performance of inseminated females held in conventional or unisex maturation systems showed no significant differences. Artificial insemination was considered a practical approach to overcome the lack of mating of F. paulensis under large-scale conditions with acceptable reproductive performance. The results also indicate the possibility of holding females separately from males, which could mean significant improvements in maturation systems through the increase in the stocking density of spawning females. [Copyright &y& Elsevier]

The Asian elephant (Elephas maximus) population in the wild has been in decline for several decades and breeding in captivity has not been self-sustaining. The use of artificial insemination (AI) can help overcome many of the difficulties associated with breeding elephants in captivity; however, the ability to store semen for extended periods of time is critical to the successful application of AI to elephants. The objective of the present study was to assess the effects of four different semen extenders and the presence of egg yolk on the viability and motility of Asian elephant semen stored at 4 °C. High quality ejaculates (n=4) were collected from two Asian elephant bulls by rectal massage. Aliquots of each ejaculate were extended in four different diluents (Beltsville thawing solution (BTS); Tris–citric acid (TCA)/fructose-based; Beltsville F5 (BF5); dextrose-supplemented phosphate-buffered saline (PBS)) with or without egg yolk then cooled and stored at 4 °C. The percentages of viable (viability) and motile (motility) sperm were evaluated at 8, 24 and 48 h following collection. The addition of egg yolk significantly reduced the percentage loss in viability from initial collection to 48 h compared to extenders without egg yolk (17.0±8.2 versus 32.6±8.9 decline in percent viable sperm in the population, respectively; P<0.05). Extender and egg yolk affected (P<0.005) total motility and percent progressively motile sperm at all evaluation times during incubation. TCA + egg yolk maintained higher (P<0.05) levels of progressive motility compared to other extenders supplemented with egg yolk. These results indicate that Asian elephant semen extended in TCA diluent supplemented with egg yolk can maintain at least 50% viability and motility when stored at 4 °C for 48 h. [Copyright &y& Elsevier]

Eggs of the Japanese eel, Anguilla japonica, were obtained by hormonally induced maturation and ovulation, and then categorized as buoyant and non-buoyant types in seawater after insemination. The water content of the buoyant eggs, 93.1%, was significantly higher than that of the non-buoyant eggs, 87.5%. Furthermore, the buoyant eggs had 1.5 times more free amino acids (FAA), 573.5 μmol/g dry weight (DW), than the non-buoyant eggs, 385.8 μmol/g DW, while no significant difference in egg lipid content or protein content was found. Compared to other pelagic eggs reported in the literature, the FAA content of the buoyant eggs was relatively low, although the water content was similar. These results suggest that there is a close relation between the quality of Japanese eel eggs obtained by typical hormonal inducement and the FAA content of the eggs, but the roles of FAA in the acquisition of egg buoyancy and fulfilling the early nutritional requirements are comparatively minor. [Copyright &y& Elsevier]

The site of semen deposition (vaginal, cervical or uterine) and the time required for transvaginal artificial insemination (AI) using the Gourley Scope method of AI vs. a standard speculum-guided AI gun was assessed in three species of farmed deer (fallow, red and white-tailed deer). Pregnancy rates using these two transvaginal AI methods were also compared in a commercial herd of fallow does. The estrous cycles of fallow (n=53), red (n=27) and white-tailed (n=27) deer were synchronized using half of a Norgestomet ear implant inserted for 14 days. Following implant removal, does were artificially fix-time inseminated with frozen-thawed semen using either a 0.5 ml standard French-style speculum-guided AI gun or the Gourley Scope method of AI. The time-frame (i.e., duration in minutes) for the AI procedures performed on each animal and the location of semen deposition was recorded. To assess effectiveness of AI, transrectal ultrasonography was conducted in fallow deer 45 days post-AI for pregnancy diagnosis. In fallow does and red deer hinds, use of the Gourley Scope (96.3 and 100%, respectively) enabled a greater (P<0.05) percentage of intrauterine inseminations compared to the speculum-guided AI gun (73.1 and 42.9%, respectively). In contrast for white-tailed does, both the speculum-guided AI gun and the Gourley Scope resulted in a similar proportion of intrauterine inseminations (92.3 and 100%, respectively). The duration for performing the two transvaginal AI techniques were also similar in red and white-tailed deer. However in fallow does, AI with the aid of the Gourley Scope took longer (P<0.05) to conduct than the speculum-guided AI gun (6.6±0.72 and 4.3±0.34 min, respectively). The pregnancy rate using both methods of AI combined was overall 32.1% for fallow does. The total percentage of fallow does that conceived with the speculum-guided AI gun was 42.3% whereas the pregnancy rate using the Gourley method of AI was only 22.2% (P<0.05). Although both AI methods may be acceptable for use in farmed deer, the standard speculum-guided AI gun is simpler and produced more pregnancies compared to the Gourley Scope method in fallow deer, despite a greater proportion of intrauterine inseminations using the Gourley Scope. [Copyright &y& Elsevier]

Abstract: Artificial inseminations using sperm masses were applied to intra-specific crosses of Penaeus (Litopenaeus) occidentalis, as validation of the technique. Reciprocal, inter-specific crosses of P. occidentalis and P. (Litopenaeus) stylirostris were performed; complementary, inseminations of both species were also accomplished with sperm masses of P. (Litopenaeus) vannamei. Intra-specific inseminations generated viable spawns with 54.2% of normal embryos; on the contrary, inter-specific spawns were all unfertile, with a high rate of eggs showing abnormal intracellular corpuscles. Based on recent discoveries about sperm maturation/capacitation, a new approach to study inter-specific sperm–egg compatibility in the sub-genus Litopenaeus is discussed. [ABSTRACT FROM AUTHOR]