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

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

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

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]

Two species of loach, the mud loach ( Misgurnus mizolepis Günther) and the cyprinid loach ( M. anguillicaudatus Cantor), are commercially important in Korea both for food and ceremonial purposes. The mud loach has superior potential for aquaculture in terms of growth, whereas the cyprinid loach has a more desirable body shape and colour. This study was conducted to produce reciprocal diploid and triploid hybrids and to evaluate their potential benefits in culture by examining growth performance, morphometrics and gonad development. Reciprocal diploid and triploid hybridization was performed by artificial insemination without or with the induction of triploidy. The successful formation of diploid or triploid karyogamy was verified by flow cytometric analysis. Body weights of induced reciprocal diploid hybrids were intermediate between those of the two parental species, i.e. hybrids were heavier than cyprinid loach but lighter than mud loach. In contrast, the growth performance of triploid hybrids was similar to that of their corresponding maternal parents. Diploid hybrids showed intermediate morphometric traits between the two species; however, the body proportions of triploid hybrids were more similar to those of the maternal species than the paternal species. Histological analyses indicated that reciprocal diploid hybrids of both sexes were able to reach maturity, as evidenced by the presence of mature oocytes or spermatozoa in the gonad tissues. However, triploid hybrids showed stringent sterility at the gonadic level; the sizes of ovaries and testes were much smaller, and gonad development was abnormal and significantly retarded. [ABSTRACT FROM AUTHOR]

Studies the reproductive cycle of the bottlenose dolphin. Characteristics of the estrous cycle; Artificial insemination using frozen-thawed spermatozoa; Excretory dynamics of urinary luteinizing hormone and ovarian steroid metabolites; Effect of a synthetic progesterone analog on reproductive hormone excretion.

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

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

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

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The silkworm, Bombyx mori, has a dimorphic sperm system. The eupyrene sperm is the sperm to fertilize eggs and the apyrene sperm plays a crucial role for assisting fertilization. Heat-treated (33°C for 96h) Daizo (DH) males, one of the strains in the silkworm, produce only eupyrene sperm, while in triploid males only apyrene sperm are functional. Though both types of males are found to be sterile, double copulation of the two males with a single female greatly increases fertility. Here we examined the fertilizing ability of eupyrene and apyrene sperm by means of an artificial insemination technique previously established in B. mori. Neither the eupyrene sperm collected from DH males, nor the apyrene sperm from triploid males have the ability to fertilize eggs. Artificial insemination with the mixture of eupyrene and apyrene sperm leveled up the frequency of fertilized eggs to more than 80%. When cryopreserved DH sperm (eupyrene sperm) were subjected to the same experiment, more than 95% fertilized eggs were obtained. These results confirmed that apyrene sperm play an important and indispensable role in fertilization in B. mori. Separate collection of functional eupyrene sperm and functional apyrene sperm and success of fertilization by means of the artificial insemination technique are applicable for further studies to elucidate the function of apyrene sperm. J. Exp. Zool. 297A:196–200, 2003. © 2003 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Examines the sperm competition mechanisms in wild bird population. Efficacy of anticopulator; Determination on the frequency and timing of extra-pair inseminations; Fertilization probability caused by female behavior.

This paper reports preliminary studies designed to evaluate hybridization through natural mating and artificial insemination between female Penaeus japonicus Bate, 1888 and male Penaeus kerathurus Forskäl, 1775. Although both species were able to mature in captivity, natural copulation was not achieved. Different techniques, including manual squeezing in the base of the fifth pair of pereiopods. electrical stimulation and dissection, were used for the artificial insemination experiments. However, only dissection gave spermatophores. Stained sperm showed no malformations and its viability was very high. Seven out of 10 inseminations (70%) produced eggs, but the development of embryos was abnormal. It is suggested that the high genetic distance between these species is responsible for these results. [ABSTRACT FROM AUTHOR]

The study was done to understand the preservation of Washed Spermatozoa of Mehsana Buck at Refrigeration temperature. The washed spermatozoa from 78 semen ejaculates, 26 each from 3 Mehsana bucks, were preserved at 4 ± 10C temperature in SM and TCFEY diluents up to 72 hours and the physical characteristics of spermatozoa were studied to assess the suitability of diluents. The individual sperm motility and live spermatozoa decreased significantly whereas abnormal spermatozoa increased significantly (P<0.05) at each 12 hrs interval of preservation in two diluents. The effect of diluents on spermatozoan characteristics was less pronounced in SM than TCFEY diluent. Among the interaction studies, stage of preservation affected the live spermatozoa and their morphology. The findings suggested superiority of SM diluent over TCFEY for preservation of Mehsana buck semen. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate the various alternations between long and short days of sunlight in an attempt to optimise the production of semen collected for artificial insemination (AI) under field conditions. The results suggest that alternation between long days and short days allowed for the production of AI doses all year round in large scale under field conditions. The alternation 45 LD / 45 SD seems to be the better treatment to optimise the production of AI doses. Under these conditions the production of AI doses was not influenced by the rhythm of collection. More investigations are necessary to optimise the rhythm of weekly collection. [ABSTRACT FROM AUTHOR]

Artificial insemination (AI) using sex-selected sperm of bottlenose dolphins is currently used for the reproductive and social management of captive populations, but distance of males to the sorting facility represents a limitation of the procedure. Sorting and recryopreservation of previously frozen–thawed (FSF) sperm would facilitate the global application of this technology. Although a calf has been produced using FSF sperm (O’Brien et al.2009 Theriogenology 71, 98–107), a comprehensive examination of the in vitroquality of such samples is needed. The objective was to compare the in vitroquality of nonsorted (CNTR) and sorted (FSF) dolphin sperm before and after recryopreservation using straw (STR) and directional freezing (DF) methods. At all assessment intervals, sperm were evaluated for 1) motility parameters with computer-assisted sperm analysis (CASA); 2) plasma membrane integrity (viability) and acrosome integrity using propidium iodide/fluorescein isothiocyanate-labeled peanut agglutinin (PI/FITC-PNA) staining and 3) DNA denaturation using the sperm chromatin structure assay (SCSA). Semen from 3 ejaculates×3 males was cryopreserved by DF. After thawing, samples were divided into CNTR and FSF. The CNTR sperm were recryopreserved using STR and DF methods with assessments performed after the first thaw (PT1) and before recryopreservation (PF2). The FSF sperm were prepared for sorting using a density gradient centrifugation (DGC) method, stained with Hoechst 33342, sorted (SX MoFlo®, Dako, Fort Collins, CO, USA), then recryopreserved using STR and DF methods. The FSF sperm were assessed post-PT1, post-DGC, post-stain, post-sort, and at PF2. After the second thaw (PT2), CNTR and FSF samples were diluted (1:0.1, vol/vol) with Androhep Enduraguard™ (AE; Minitube of America, Verona, WI, USA), incubated at room temperature, and assessed at 0, 6, 12, 18, and 24h PT2. The PT1 samples retained high proportions of their PF1 total motility (TM) and progressive motility (PM) (mean±SD; 87.9±7.3% and 92.2±5.9%, respectively). The FSF sperm had improved (ANOVA; P<0.05) motility (TM, PM, VAP, VCL, VSL) and viability at PF2 compared with PF1. The FSF sperm recryopreserved using DF had higher (P<0.05) motility over the 24-h post-thaw incubation period compared with STR. The CNTR sperm DNA fragmentation remained unchanged throughout the process. The DNA fragmentation of FSF samples increased after staining (P<0.05), then decreased during the PT2 incubation period, stabilising at lower values (P<0.05) than CNTR from 6 to 24h PT2. This unusual pattern indicates a possible interaction between Hoechst 33342 and acridine orange. After recryopreservation, the viability of FSF sperm was higher (P<0.05) than that of CNTR sperm. Results indicate that bottlenose dolphin sperm undergoing cryopreservation, sorting, and recryopreservation are of adequate quality for use in AI. [ABSTRACT FROM AUTHOR]

The reproductive physiology of the Pacific white-sided dolphin, Lagenorhynchus obliquidens, was characterized to facilitate the development of artificial insemination (AI) using cryopreserved spermatozoa. Specific objectives were to: 1) describe reproductive seasonality of the Pacific white sided dolphins; 2) describe urinary LH and ovarian steroid metabolites during the estrous cycle; 3) correlate LH and ovarian steroidal metabolite patterns to ultrasound-monitored follicular growth and ovulation; and 4) assess the efficacy of synchronizing estrus, sperm collection/cryopreservation, and intrauterine insemination. Ovulations (64%, n=37) and conceptions (83%, n=18) occurred from August to October. Peak mean serum testosterone (24 ng/ml), cross-sectional testicular area (41.6 cm²), and sperm concentration (144.3x107 sperm/ml) occurred in July, August, and September respectively. Spermatozoa were only found in ejaculates from July to October. Estrous cycles (n=22) were 31 d long and were comprised of a 10 d follicular and 21 d luteal phase. Ovulation occurred 31.2 h after the onset of the LH surge and 19.3 h after the LH peak. Follicular diameter and circumference within 12 h of ovulation were 1.52 and 4.66 cm respectively. Estrus synchronization attempts with altrenogest resulted in 17 (22%) ovulatory cycles with ovulation occurring 21 d post-altrenogest. Ten AI attempts using cryopreserved semen resulted in five pregnancies (50%). The mean gestation length was 356 days (range 348-367). These data provide new information on the Pacific white-sided dolphin's reproductive physiology and collectively enabled the first application of AI in this species. [ABSTRACT FROM AUTHOR]

In addition to gametes, mammalian internal fertilisation has required the evolution of assorted anatomical, physiological and biochemical devices to deal with intra- and inter-sexual conflict such as sperm competition and female cryptic choice respectively. The seminal coagulum of primates and other mammals is viewed as one of such devices. Among primates, the seminal coagulum characteristically occurs in multi-male and multi-female species, leading us to suppose that it intervenes in sperm competition. However, it can also provide cues to the female reproductive tract about male desired or undesired traits, and therefore deter or favour sperm survival and migration. The present work investigates whether the seminal coagulum of the black-handed spider monkey enhances sperm fertilisation chances by improving the female reproductive tract conditions, and if the female reproductive tract is 'blind' to semen or behaves selectively towards ejaculates of different males. A series of artificial inseminations were done in five females, using the ejaculates of three different males, one at a time, and measuring the presence of distinct types of sperm inside the uteri at 10, 30 and 60 min following the insemination. The presence of coagulum, menstrual phase, and male and female identity only affected fast, straight-moving sperm, with larger amounts of fast sperm appearing inside the uteri when ejaculates had seminal coagulum, as well as when in the periovulatory phase. There was great intra-uterine fast-sperm variation regarding which male's semen inseminated which female. The results provide evidence to account for sexual conflict in the spider monkey as well as a methodological approach to this kind of study. [ABSTRACT FROM AUTHOR]

The purpose of this investigation was to develop a non-invasive technique to artificially inseminate snakes using the corn snake, Elaphe gutatta, as the model representative for this taxon. Semen was collected by first applying pressure to the lower abdomen in a continuous distal motion toward the cloaca to remove any feces or urates. The cloaca was then gently washed using phosphate-buffered saline, and a more localized pressure was applied to each side of the vent to evert the hemipenes and, subsequently, the ejaculate. The semen was collected using a sterile transfer pipette and placed into 70 to 90 L of medium (TL-HEPES solution; Cambrex Bio Science, Inc., Baltimore, MD, USA04–616F) in a sterile microcentrifuge tube, and then analyzed for overall motility, rate of forward progression (RFP, 0–5), and concentration. Based on a previously reported procedure, 10 females were inseminated with either fresh (n = 5) or cooled semen (n = 5; refrigerated for 3 days) one week after recovering from a hibernation period required to stimulate reproduction in this species. The overall sperm motility and concentration for females inseminated with fresh or cooled semen was 92%, 9.6 million sperm mL-1; and 85%, 6.1 million sperm mL-1, respectively. Immediately prior to insemination, the same method for expressing feces and urates in the males was applied to the females. The insemination dose (50 L semen per oviduct) was drawn into a 1-mL latex- and silicone-free tuberculin syringe (Norm-Ject; VWR, Batavia, IL, USA) that was connected to a feeding/dosing needle (EJAY International, Issaquah, WA, USA) with a ball tip to prevent any potential damage during the insemination. The tip of the needle was then moved around the inner tissue of the vent to relax the cloaca, and the insertion continued until resistance was found indicating the vicinity of the oviducts. The extended semen was carefully deposited on both sides, and then the needle was slowly withdrawn. The offspring were tested for parentage to verify the success of the insemination. Blood was collected from the dorsal aorta posterior to the cloaca and stored in 10 mM Tris at 4C. The DNA was extracted using a phenyl : chloroform : isoamyl alcohol (PCI) extraction method. Eight microsatellite loci were used for the paternity exclusion analysis: Eob1, Eob3, Eob10, Eob13, Eob16, Eob34, Eob366, and Eob373 (IDT, Coraville, IA, USA). All males and females in the collection were tested, and parental candidates were excluded if 2 or more allele mismatches occurred. From the total number of females inseminated, 3 females laid 51 eggs. Two females inseminated with cooled semen laid 36 eggs, of which 5 eggs were hatched, and the remaining were either unfertilized (n = 25) or non-viable (n = 6). All 5 hatched eggs were laid by one of the females. The third female inseminated with fresh semen laid 15 eggs, resulting in 5 hatching and 10 unfertilized eggs. The parentage test validated the AI a success as the alleles correlated between the adults and the offspring. In conclusion, artificial insemination was successful using both freshly collected and cooled (3 days) semen. Further studies are underway to improve the success rate in order to maximize the efficiency of this technology, and thus assist in the genetic preservation of endangered snake species. [ABSTRACT FROM AUTHOR]

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