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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]

The present study was carried out to investigate the embryonic and larval development of freshwater catfish Rita rita. The mature eggs and sperms were collected by using artificial insemination technique and fertilized eggs were incubated in mini circular hatchery with provision of continuous water supply. The fertilized eggs were transparent, demarsal, spherical, non-adhesive and brownish in colour with a diameter ranging between 1.3 to 1.6 mm. First cleavage occurred within 25-30 min post-fertilization at temperature of 28±1°C. Hatching started 22 h post-fertilization and completed within 24 h at the same temperature range. Newly hatched larvae were 2.0 mm in length devoid of mouth and pigmentation and started feeding within 48-60 h post-hatching. To date, this is the first time the early embryonic and larval development of freshwater catfish R. rita is described. Thus the findings of the present study provide valuable information that may help establishing the large scale seed production technique of Rita. [ABSTRACT FROM AUTHOR]

Contents [ABSTRACT FROM AUTHOR]

For some endangered parrot species, captive breeding may be the only insurance for their survival. However, many individuals in captivity do not reproduce. Artificial insemination (AI) may help overcome reproductive failures or geographic distance. For semen collection in birds, massage is the most commonly used method. However, this process, which usually requires capture and restraint, involves risk of stress and injuries. The aim of this experiment was to train large macaws to accept the physical manipulations of their body parts needed for the artificial insemination process. Within 15 weeks, a male and a female Buffon's macaw (Ara ambiguus) learned to accept handling without apparent stress. A pair of green-winged macaws (Ara chloropterus) progressed more slowly and displayed some signs of stress. This stress highlights the need to monitor the birds' possible signs of discomfort during the training in order to adapt the working protocol. These results demonstrate that it is possible to train the AI behaviors, thus avoiding the capture, restraint, and anesthesia. An added benefit to this is the potential for the training to provide a form of behavioral enrichment. [ABSTRACT FROM AUTHOR]

Biodiversity is the basic indicator, which expresses the durability and stability zone in direct relationship with the environment and life. Biodiversity monitoring both quantitatively and qualitatively puts us in contact with reality environment and its stability, because biodiversity is constantly changing. Only by measuring the biodiversity we can perceive the sudden chances, which influence directly our life in order to interfere with redesigning the media by ecological reconstruction. The application of biotechnologies to buffalo breeding is paramount to remedy the existing situation. The most numerous Romanian buffalos are the Carpathian type, with valuable gene resource, well adapted to the cold climate for buffalo. Results of artificial insemination in this species are influenced by the quality of semen used. We know that there are periods in which sperm are immobile, so it is important to test the ability of sperm fertilized. This test can be performed in vitro using direct or indirect methods. Utilization of Polamski scale principle remains present, but can be improve and combine with Milovanov resistance and "in vitro" capacitating methods. For determination of fertilizing power we use "in vitro" capacitation methods: direct method, indirect method and "swim-up" technique who are obligatory in E.U. We use this tests because Polamski scale can't be applied, knowing that an immobility of buffalo semen in some conditions is a characteristic of this semen, semen that can be recovery on the normal parameters. Milovanov resistance test have restricted efficiency because NaCl solution have a negative effect over the sperms. Semen charges testing with these new methods they present a good fertilizing power. This test is easier to realize and takes of decision is faster. [ABSTRACT FROM AUTHOR]

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]

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: 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]

Clinical and paraclinical investigations (morpho-pathologic, bacterioscopic, bacteriologic and sensitiveness test to antibodies) done on 20 fish (rainbow trout), dead or in agony state, taken from a furunculosis pesthole in Salmonidae and proceeded from an intensive exploitation unit of trout, in Cluj county, put in evidence the following aspects :To clinical exam, in breeding basins, it appears individuals (5 %) which have presented locomotor troubles, listless and untidy swimming in water surface, lateral swimming anorexia, and on tegument it is observed diffuse hemorrhagic zones, lost of fish scale and necrosis of fins. It appears also secondary infections with fungi of Saprolegnia genus under the form of a white- dirty, downy film. From the 20 trut corpses were isolated 2 bacterial strains, from anterior kidney and from raclage in fin basis, with congestive lesions. Bacteria isolated are developing only after an incubation at 20 - 25° C and does not grow up to 37° C, and to microscopic exam directly from sauce, the mobility is absent Classic biochemical tests put in evidence the following positive biochemic properties: cataloze, oxidaze, indolal and the presence of hemolytic activity. Biochemical properties testing on API20E gallery also puts in evidence properties as : positive gelatinousis and sucraze fermentation. Based on bacterial strains development isolated only to 20 - 25 °C and the above mentioned biochemic properties, respectively the absence of pigment, bacterial strains isolated are appointed in Aeromonas genus, Aeromonas salmonicida species, achromogenes subspecies. By sensitiveness test in different medicine substances, using antibiogram technique, bacterial strains isolated from pesthole are sensitive in a decreasing order to: nalidixic acid, oxitetracycline, florfenicol and eurofloxacin and resistant to ampicyline, amoxiclav and colistin. [ABSTRACT FROM AUTHOR]

In a microchromosome-carrying laboratory stock of the normally all-female Amazon molly Poecilia formosa triploid individuals were obtained, all of which spontaneously developed into males. A comparison of morphology of the external and internal insemination apparatus and the gonads, sperm ploidy and behaviour, to laboratory-bred F hybrids revealed that the triploid P. formosa males, though producing mostly aneuploid sperm, are partly functional males that differ mainly in sperm maturation and sexual motivation from gonochoristic P. formosa males. [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]

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]

The role of monovalent (Na+, K+) and divalent (Ca2+, Mg2+) cations in Bufo arenarum fertilization was analysed. Our results showed that the highest fertilization percentages were obtained when strings of uterine oocytes (UO) were inseminated. Under these conditions, full jelly (FJ), which represents the jelly coats surrounding the oocytes at the time of deposition, contained 68.5 ± 7.0 mM Na+, 27.4 ± 2.4 mM K+, 6.3 ± 0.9 mM Ca2+ and 6.9 ± 0.9 mM Mg2+. When the strings of oocytes were washed in deionized water, these cations diffused into the liquid medium surrounding them. There was a marked similarity between the loss of Ca2+ in the jelly and the decrease in the fertilizability of the UO. Furthermore, the use of chelating agents of divalent cations showed the importance of the Ca2+ contained in the jelly. When Ca2+ was sequestered from the jelly coats by the addition of the chelating agents to the insemination medium as well as by pretreatment of the UO strings, a decrease in fertilization percentages occurred, this effect being dose dependent and more markedwith EGTA. These results demonstrate that the Ca2+ in the jelly plays a role in fertilization. Nevertheless, taking into account that during the washing of the jelly other jelly coat components were diffused and considering that the addition of Ca2+ to the insemination medium reverted significantly, but only partially, the loss of fertilizability of jellied UO (washed), the participation of other components in the fertilization mechanism is suggested. [ABSTRACT FROM AUTHOR]

Copyright of CES Medicina Veterinaria & Zootecnica is the property of Universidad CES and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

The present study investigated the embryonic and larval development of Indian catfish, Heteropneustes fossilis from fertilization until metamorphosis. The fully matured eggs and sperm were obtained by artificial insemination. The fertilized eggs were demersal, nonadhesive, spherical, and brownish green in color. The average diameter of the fertilized eggs ranged from 1.30 to 1.50 mm. The incubation period was from 23-24 h at an average temperature of 29±1°C. The newly hatched larvae were 2.5±0.2 mm in length. The yolk absorption was completed within three d after hatching. The aerial breathing behaviour of the larvae was observed 10 d after hatching. The larva resembled the adult in its external features and was metamorphosed to young juveniles within 20 d post-hatching. [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]

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.

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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]

The blackbuck (Antilope cervicapra) is a small (20–30 kg) Indian antelope that is listed on Schedule I of the Indian Wildlife Protection Act, 1972. Studies were undertaken to develop assisted reproductive technologies, such as synchronisation of oestrus and non-surgical AI, to support the conservation and genetic management of this Indian antelope. Semen characteristics, testosterone levels and the feasibility of short-term cold storage of semen were investigated. Furthermore, different oestrous synchronisation protocols (norgestomet implants and prostaglandin injections) were evaluated for successful AI, defined as the birth of live young. Norgestomet ear implants and i.m. administration of pregnant mare's serum gonadotropin (PMSG) resulted in successful pregnancies in two of five inseminated females, but both had twin pregnancies that were delivered prematurely. In contrast, two injections of prostaglandin 11 days apart were effective in synchronising oestrus in the blackbuck. Transcervical AI in oestrous-synchronised animals 72 and 96 h after the second prostaglandin injection resulted in successful pregnancies in four of six inseminated females (67%) and resulted in the delivery of three live fawns. These studies demonstrate the potential application of AI technology for the conservation of endangered ungulates. To our knowledge, this is the first report regarding the synchronisation of oestrus and successful non-surgical AI in blackbuck. [ABSTRACT FROM AUTHOR]

A unique feature of the reproductive physiology of Asian elephants (Elephas maximus) is the occurrence of two LH surges before ovulation, instead of one. An anovulatory LH (anLH) surge, the function of which is unknown, occurs consistently 3 weeks before the ovulatory LH (ovLH) surge that induces ovulation. Thus, the ability to induce an ovLH surge would be useful for scheduling natural mating or artificial insemination. The present study tested the efficacy of a gonadotrophin-releasing hormone agonist (GnRH-Ag) to induce LH surges during the follicular phase of the oestrous cycle, which resulted in varied LH responses, but generally none were as high as previously documented natural surges. Thus, for the ovulation-induction trials, nine females were administered 80 μg GnRH-Ag intravenously at three time periods during the oestrous cycle, namely the anovulatory follicular phase, the ovulatory follicular phase and the luteal phase. During the late anovulatory follicular phase, nine of 10 females (90%) responded with an immediate LH surge followed 15–22 days later by an ovLH surge or a post-ovulatory increase in progestagens. In contrast, despite responding to the GnRH-Ag with an immediate increase in LH, none of the females treated during other periods of the oestrous cycle exhibited subsequent ovLH surges. One cow got pregnant from natural mating following the induced ovLH surge. In conclusion, ovLH induction is possible using a GnRH-Ag, but only during a specific time of the anovulatory follicular phase. [ABSTRACT FROM AUTHOR]

The cytological process of induced gynogenetic development and subsequent chromosome duplication by a cold shock treatment was observed in Japanese flounder Paralichthys olivaceus (Temminck et Schlegel). Mature eggs were at the metaphase of the second meiosis when inseminated with ultraviolet (UV)-irradiated sperm of red sea bream Pagrus major. After the beginning of cold shock treatment, the previously visible spindle became invisible, probably due to the side effect caused by cold shock treatment. The chromosomes at the centre of the metaphase plate were condensed. This condition continued during the duration of the cold shock treatment and several minutes after it. The release of the second polar body was blocked and it developed into a female-like pronucleus. Then, it fused with the female pronucleus to generate a diploid zygotic nucleus, and the egg exhibited the first mitosis. Consequently, the haploid female chromosome set of the egg was doubled by the inhibition of the second polar body release. There was a significant delay in developmental time in the gynogenetic eggs when compared with that in the normal eggs. From the time of insemination to early cleavage, the UV-irradiated heterospecific sperm nucleus remained condensed. [ABSTRACT FROM AUTHOR]

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]

The populations of the only known self-fertilizing vertebrate Kryptolebias marmoratus (Poey, 1880) (formerly known as Rivulus marmoratus Poey, 1880; Cyprinodontiformes: Aplocheilidae) usually consist of different homozygous lineages; however, heterozygous individuals are found occasionally and ratios of homozygosity and heterozygosity in populations are dependent on the proportion of males. However, it is still unclear what impact male-mediated genetic diversity has on the phenotype of K. marmoratus. To clarify this, we attempted outcrossing between male and hermaphrodite of two different clonal strains with different life-history traits using artificial insemination, and examined the genotypes and growth of the hybrid F2 generation. We detected genetic differences between the two clonal strains using amplified fragment length polymorphism (AFLP) analysis with 3 primer combinations, and then obtained 11 AFLP markers. From a total of 31 artificial inseminations with two clonal strains, 1 of 13 hatched fish clearly indicated heterozygosity. The hybrid F2 generations were also heterozygous. Moreover, the growths of the hybrid F2 generation were intermediate of the parental strains from days 0 to 30. Therefore, outcrossing changes genetic architecture and the new genotypes potentially result in new phenotypes of the subsequent generations of K. marmoratus. It may also play a role in adaptation to new environments and the facilitation of local adaptation. Les populations du seul vertébré connu capable d’autofécondation Kryptolebias marmoratus (Poey, 1880) (désigné antérieurement Rivulus marmoratus Poey, 1880; Cyprinodontiformes : Aplocheilidae) consistent généralement en différentes lignées homozygotes; cependant, on trouve à l’occasion des individus hétérozygotes et les rapports de l’homozygotie et de l’hétérozygotie dans les populations dépendent de la proportion de mâles. Cependant, il n’est pas clair quel impact la diversité génétique causée par les mâles a sur le phénotype de K. marmoratus. Afin d’éclaircir la question, nous avons tenté à l’aide de l’insémination artificielle de faire des croisements exogames entre des mâles et des hermaphrodites de deux différentes souches clonales possédant des traits de cycle biologique différents; nous avons ensuite examiné les génotypes et la croissance de la génération hybride de F2. Nous détectons des différences génétiques entre les deux souches clonales à l’aide d’une analyse du polymorphisme de la longueur des fragments amplifiés (AFLP) avec une combinaison de 3 amorces et nous obtenons 11 marqueurs AFLP. Dans un ensemble de 31 inséminations artificielles avec les deux souches clonales, 1 des 13 poissons éclos montrait des signes évidents d’hétérozygotie. Les générations hybrides de F2 sont aussi hétérozygotes. De plus, la croissance de la génération hybride de F2 est intermédiaire entre celles des souches parentales des jours 0 à 30. Ainsi, les croisements exogames changent l’architecture génétique et les nouveaux génotypes produisent potentiellement de nouveaux phénotypes dans les générations subséquentes de K. marmoratus. Ils peuvent aussi jouer un rôle dans l’adaptation aux nouveaux environnements et l’amélioration de l’adaptation locale. [ABSTRACT FROM AUTHOR]

Ejaculates were collected from a beluga (Delphinapterus leucas) to gain an understanding of sperm biology and develop a short-term sperm preservation method for use in artificial insemination (AI). Ejaculate parameters and biochemistry, semen production and serum testosterone concentrations of an adult male were characterised for 21 months. Sperm viability, acrosome integrity and morphology did not change (P> 0.05) but ejaculate volume, sperm concentration and total spermatozoa per ejaculate were higher (P< 0.05) from January to June than from July to December. Peak testosterone concentrations (P< 0.05) were observed from October to April (8.0 ± 1.6 ng mL-1). The effects of hyaluronic acid (HA), antioxidants, storage temperature and time on in vitrosperm characteristics were examined. Motility parameters and viability were improved (P< 0.05) when semen was stored at 5°C compared with 21°C. During the first 24 h of storage sperm agglutination was absent only at 5°C in the presence of HA. A nulliparous 28-year-old female was inseminated endoscopically with liquid-stored semen. A pregnancy and birth of a calf was achieved following AI for the first time in this species, thereby validating both the AI technique and the fertility of beluga spermatozoa after chilled storage in a specialised diluent. [ABSTRACT FROM AUTHOR]

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]

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]

Contents Over the last few decades, rhinoceroses and elephants became important icons in the saga of wildlife conservation. Recent surveys estimate the wild Asian ( Elephas maximus) and African ( Loxodonta africana) elephant populations to be, at most, 50 250 and 637 600 respectively. For the five rhinoceros species, black ( Diceros bicornis), white ( Ceratotherium simum), Indian ( Rhinoceros unicornis), Javan ( Rhinoceros sondaicus) and Sumatran rhinoceros ( Dicerorhinus Sumatrensis), the population estimates of 3610, 11 330, 2400, 60 and 300, respectively, are of even greater concern. Protected against habitat loss, poaching and left undisturbed, rhinoceros and elephants reproduce well in the wild. But small and decreasing populations make successful captive management of these taxa increasingly important. In captivity, however, most populations face possible ‘extinction’ because of historically poor reproductive performance. From the first descriptions of the reproductive anatomy and the oestrous cycle ( Laws 1969 ; Kassam and Lasley 1981 ; Balke et al. 1988a,b ; Plotka et al. 1988 ; Godfrey et al. 1991 ) to the present use of advanced assisted reproduction technologies, researchers have strive to understand the function and dysfunction of the reproductive biology of these charismatic species. This paper reviewed the current knowledge on rhinoceros and elephant reproduction biology, reproductive cycle, gestation, dystocia, reproductive pathology, oestrous induction and artificial insemination, sperm sexing, IVF and contraception, and how this knowledge is or might be used to aid species conservation for maximal reproductive efficiency and enhancement of genetic management. [ABSTRACT FROM AUTHOR]

Clearnose skates, Raja eglanteria, are a common species of skate found seasonally along the Atlantic coast of North America from Cape Cod to mid-Florida and in the Gulf of Mexico from mid-Florida to eastern Texas. Clearnose skates inhabit the west central coast of Florida during winter months, when Gulf temperatures are conducive to mating and egg-laying (approximately 16°C–22°C). Specimens collected during this time will breed in captivity and, if maintained at 20°C, mated females will store sperm and continue to lay fertile eggs for up to six months. With fertile eggs available from day of laying until hatching, the first complete description of batoid elasmobranch embryonic development with a timetable has been established and is presented here. If eggs are maintained at 20°C, appearance and progression of embryonic events proceed over a reproducible timeline. The timeline has been divided into discrete time periods into which developmental events have been grouped. These include cleavage and expansion of the blastodisc (day 1–day 4), embryonic axis and neural tube formation (day 4–day 7), pharyngeal pouches and gill filament development (day 10–day 28), expansion of fins and transition from external filaments to internal gills (week 4–week 7), and epidermal pigmentation and yolk absorption (week 8–week 12). Fully formed offspring hatch after an incubation period of approximately 12 weeks (85 ± 6 days). To examine possible mechanisms of transport and storage of sperm in the reproductive tract of females following copulation, experiments resulting in the first artificial insemination for any elasmobranch fish are described. These experiments provide evidence for functional roles of the alkaline gland, whose secretions stimulate sperm motility and may enhance migration of sperm to the oviducal glands, and the clasper gland, whose secretions may provide nutrition during storage of viable sperm in the oviducal gland. [ABSTRACT FROM AUTHOR]

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]

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]

The freshwater tolerance of starry flounder Platichthys stellatus, stone flounder Kareius bicoloratus and their reciprocal hybrids, produced by artificial insemination, were examined in the larval, juvenile and immature phases. Survival rate on being transferred to fresh water in the pre-settlement phase was 0% in stone flounder and hybrids and 16·7% in starry flounder. This rate in the post-settlement phase was elevated to >50% in starry flounder and hybrids but was still 0% in stone flounder and similarly in the immature period starry flounder and hybrids survived in fresh water, although stone flounder did not. The lamella chloride cells of the gill epithelium increased in starry flounder and hybrids in fresh water in all periods. Densities of lamella chloride cells increased from 1·6 ± 0·4 (mean ±s.e. number of cells per 1 mm filament) before the transferral (day 0) to 60·3 ± 6·2 on 14 days after the transfer to fresh water (day 14) in starry flounder in the immature period. These densities in hybrids were 0·6 ± 0·3 and 1·0 ± 0·3 on day 0, and, 35·3 ± 2·8 and 23·2 ± 4·6 on day 14, respectively. Stone flounder did not show a substantial change in chloride cell densities throughout the experimental period. These results suggest that low salinity tolerance was well developed in the settlement period in starry flounder and hybrids, and hybrids were also adapted to fresh water sufficiently regardless of the cross type. [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]

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]