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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]

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 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: 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]

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]

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]

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

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

The structure of the seminal receptacles and associated accessory pouches in the reproductive tracts of four species of Australian ovoviviparous peripatopsids is described to identify features that may relate to their inferred mode of insemination. Euperipatoides rowelli is a dermal inseminator whereas Phallocephale tallagandensis, Ruhbergia bifalcata and Cephalofovea clandestina, species with head structures in males, are considered to be vaginal inseminators. Contrary to our expectations, E. rowelli, R. bifalcata and C. clandestina have very similar seminal receptacles and display two types of accessory pouches: funnel accessory pouches that open to the haemocoel and spherical accessory pouches in which destruction of excess sperm occurs. On the other hand, Ph. tallagandensis differs in the form of its accessory pouches. Although these pouches have no direct opening into the haemocoel, sperm were observed in the act of directly penetrating their walls to gain entry from the haemocoel. Hence, despite differences in the form of their accessory pouches, all species display features that provide the potential for entry of sperm into the female reproductive tract from the haemocoel. This finding has caused us to reconsider our initial assumptions of vaginal and dermal insemination as mutually exclusive reproductive strategies. Indeed, we propose that species of peripatopsids with head structures in males that were considered to be obligate vaginal inseminators may in fact be facultative, with vaginal insemination of sexually immature females when their uterus is short and free of developing embryos followed by dermal insemination to maintain the level of sperm in storage in the seminal receptacles. This study has also provided morphological evidence for mechanisms that may facilitate female control of paternity. [ABSTRACT FROM AUTHOR]

Contents Over the past decade, there has been increasing interest in the application of reproductive technology to the conservation and management of natural populations of deer. The application of assisted reproduction technologies within natural population of deer is in its infancy. However, its future potential is enormous, particularly in relation to genetic management or conservation. This paper reviews the present state of such technologies for a wild subspecies of red deer, the Iberian red deer ( Cervus elaphus hispanicus), by discussing the major components of oestrous synchronization, semen collection/cryopreservation and insemination techniques. In addition, findings made during the course of studies on natural populations have enormous potential for the understanding of novel reproductive mechanism that may not be uncovered by livestock or human studies. A summary of these results are also reviewed here. [ABSTRACT FROM AUTHOR]

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

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]

In December 2009, 8 female Himalayan tahrs (Hemitragus jemlahicus) were synchronized by inserting CIDR devices for 13 or 15 days. One day before removal of the CIDRs, a combination of pregnant mare serum gonadotropin (PMSG) 400 IU and hCG 200 IU (PG600®, Intervet, Boxmeer, the Netherlands) was intramuscularly injected into each animal and PGF2αwas injected at the time of CIDR withdrawal. Semen was collected by electrical stimulation, diluted, cooled to 5°C for 2h, and maintained in a refrigerator before performing AI twice. Pregnancy diagnosis was conducted by Doppler ultrasonography on March 2, 2010. Four female Himalayan tahrs were impregnated, showing a fetus present in their uteri. In the first experiment in which CIDRs were inserted into the vagina of 4 Himalayan tahrs for 13 days, 3 of the tahrs were transcervically inseminated with fresh semen, whereas 1 was inseminated with frozen–thawed semen 33h after CIDR removal. As a second insemination experiment, 46h after CIDR removal, all 4 tahrs were inseminated with diluted (1:3) fresh semen. While the tahr that was later inseminated with frozen–thawed semen did not become pregnant, the other 3 Himalayan tahrs became pregnant. In the second experiment, CIDR devices were inserted into the vaginas of 4 Himalayan tahrs for 15 days. Extended (1:7)-chilled semen was used for transcervical insemination 42 and 49h after CIDR withdrawal. When performing the second insemination, it was difficult or impossible to penetrate cervical canals. Thus, only 1 tahr became pregnant. These findings show 13 days of CIDR insertion, the dose of PMSG 400 IU and hCG 200 IU, and the use of PGF2αas beneficial for inducing oestrus synchronization, and prove that extended-chilled semen may also be used for artificial breeding of Himalayan tahrs. This is the first report of successful pregnancy induced by artificial insemination of fresh or extended-chilled semen in oestrus-synchronized Himalayan tahrs. [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]

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]