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The aim of this study was to induce superfetation in hares. On day 37 or 39 of pregnancy, female hares were treated with GnRH analogue and artificially inseminated with fresh semen. Ultrasonographic examination showed that pregnant females had follicles on the ovary on 37 days of pregnancy. After a few days from insemination performed during pregnancy, all females gave birth to healthy young, and one of the females inseminated on day 37 of pregnancy gave birth to two healthy young 42 days after insemination performed during pregnancy (39 days post first delivery). The same female in the next year gave birth to two healthy young 43 days after second insemination that was performed on the day 37 during pregnancy. The obtained results confirm that superfetation is possible to occur in hares. [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: 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]

Generally natural service is preferred over Artificial Insemination (A.I.) for breeding buffaloes in India. This coupled with lack of recording for productive and reproductive traits in field conditions becomes a major handicap in implementing genetic improvement programs in buffalo. BAIF Development Research Foundation -a NGO working for improvement of rural buffalo has been implementing buffalo A.I. program over more than a decade in the state of Uttar Pradesh (which has 25% buffalo population of India). In the present study >11000 daughters of 12 sires used in AI were identified and tagged at farmers' doors. The blood samples of 9775 daughters were collected and DNA was isolated. All the 12 sires used in the buffalo improvement program and their 9775 daughters were genotyped at 10 highly polymorphic microsatellite loci distributed throughout the genome. The resultant genotype at the 10 microsatellite loci of all the sire and their daughters were compared using a small software "Confirm Paternity" developed for the purpose. If the genotype of sire and its daughters matched atleast at 8 microsatellite loci it was assumed that the daughter belonged to the recorded father. A concession of 2 mismatches were permitted due to inherent technical reasons (addition of extra a by Taq polymerase in some of the PCR products and also the sizing of the allele is never a whole number). Out of 9775 daughters of these 12 sires, only 930 failed to meet the criterion while 678 daughters belonged to one of the 12 AI sires but had wrong recording of the paternity. This revealed a correct paternity of 83% under field conditions. Based on the findings, it was concluded if carefully implemented, farmers are willing to accept and rely on A.I. for buffalo improvement and it is possible to implement buffalo field progeny testing under Indian rural field conditions for their genetic improvement. [ABSTRACT FROM AUTHOR]

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]

The article presents a study which examines the application of an artificial insemination approach in the early embryonic development of the mangrove sesarmid crab Perisesarma bidens. For the study, researchers analyzed crabs in nature exhibiting courtship behaviors at the Manko mangrove swamp in Okinawa Island, Japan. Researchers removed the sperms from the spermatheca of the similar female and diluted them in 80 percent filtered sea water (FSW). Findings suggest that the crab fertilization is of the moderately internal kind that happens in the abdominal cavity or flaps.

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: 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: 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]

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]

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]

The eel has long been esteemed as an important food fish in the world, especially in Japan, and has been used as an experimental fish for many fields of fish physiology. However, the decreases in eel resources have been a serious concern in recent years. The catches of glass eels as seedlings for aquaculture have shown a long-term decrease in both Europe and East Asia. To increase eel resources, the development of techniques for artificial induction of maturation and spawning and rearing their larvae have been eagerly desired. Recent progress of reproductive physiology of fish, especially mechanisms of oocyte maturation and ovulation in female and of spermatozoa maturation in male, facilitate to establish techniques for hormonal induction of maturation and spawning in sexually immature eels. With persistent effort to development of rearing techniques of larvae, we have first succeeded to produce glass eel. These applied techniques are may contribute to understand the basic reproductive physiology of the eel. [ABSTRACT FROM AUTHOR]

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]

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]

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 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]

Most of the progress in female great ape reproduction has focused on monitoring ovarian and endocrine activity. Perhaps of more importance has been the gradually evolving interest and willingness to consider biotechnology as a potential, viable approach for enhancing reproductive performance. Artificial insemination (AI), in vitro fertilization (IVF), and embryo transfer offer an array of possibilities for combating infertility and understanding the fundamental differences and similarities among great ape species. Multidisciplinary efforts have assessed reproductive competence, from the simple (i.e., perineal tumescence and urinary occult blood in chimpanzees) to the complex (i.e., ovum recovery and IVF following exogenous hormone treatment in chimpanzees and gorillas). Hysterosal-pingography and laparoscopy have been used to determine uterotubal patency and to identify pathological conditions in gorillas. Assays for steroid metabolites in serial urine samples, to permit accurate assessments of ovarian cyclicity. have been developed and validated for all great apes species. Stimulation of follicular recruitment and maturation h4s been achieved following administration of clomiphene citrate (chimpanzees and gorillas) and human menopausal gonadotropin (gorillas), Clomiphene citrate and human chorionic gonadotropin have been used to regulate ovulation for the Al of chimpanzees. Pregnancies have resulted in chimpanzees and gorillas following Al using fresh and cryopreserved/thawed semen; however, conception rates vary. Embryos have been nonsurgically recovered from chimpanzees after timed matings and follicular oocytes have been recovered from chimpanzees and gorillas by transabdominal laparoscopy after exogenous hormone treatment. [ABSTRACT FROM AUTHOR]

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]

In vitro fertilization of follicular oocytes harvested from ovaries and matured in vitro was attempted for 55 minke whales (Balaenoptera acutorostrata) captured for Japanese research purposes in the Antarctic Ocean during the period from November 1995 to March 1996. In Experiment 1, effects of culture duration (96 h or 120 h) on maturation of follicular oocytes and addition of caffeine (5 mM) and/or heparin (100 mu g/ml) on sperm penetration and pronuclear formation were investigated. Spermatozoa recovered from the vasa deferentia of four mature males were diluted (5-fold) and frozen at -80 deg.C. The post-thawed and pooled spermatozoa were used for in vitro insemination. A higher (P < 0.05) proportion of the oocytes cultured for 120 h (34.2%of 260) progressed beyond the second metaphase stage than of the oocytes cultured for 96 h (26.0% of 262). For the matured oocytes, higher rates of penetration (P < 0.05) and pronuclear formation (P < 0.01) were obtained in the oocytes cultured for 120 h (55.1% and 40.4%) than in those cultured for 96 h (32.4 % and 20.6916). Addition of caffeine and heparin did not show a significant effect. In Experiment 2, follicular oocytes matured for 120 h and then inseminated were cultured to examine the subsequent development in two culture systems (with and without co-cultured cumulus cells). Of 448 inseminated oocytes, cleaved embryos (2--16 cells) were observed with (5.8%) and without (4.9%) co-cultured systems. No cleavage was observed in 54 ova without insemination. These results indicate that in vitro fertilization of minke whale in vitro matured follicular oocytes with cryopreserved spermatozoa is possible, yielding cleaved embryos." [ABSTRACT FROM AUTHOR]