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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: 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: 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: 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: Short-term storage and cryopreservation of sperm are two common procedures in aquaculture, used for routine practices in artificial insemination reproduction and gene banking, respectively. Nevertheless, both procedures cause injuries affecting sperm motility, viability, cell structure and DNA stability, which diminish reproductive success. DNA modification is considered extremely important, especially when sperm storage is carried out with gene banking purposes. DNA damage caused by sperm storage is not well characterized and previous studies have reported simple and double strand breaks that have been attributed to oxidative events promoted by the generation of free radicals during storage. The objective of this study was to reveal DNA fragmentation and to explore the presence of oxidized bases that could be produced by oxidative events during short-term storage and cryopreservation in sex-reversed rainbow trout (Oncorhynchus mykiss) spermatozoa. Sperm from six males was analyzed separately. Different aliquots of the samples were stored 2h (fresh) or 5 days at 4°C or were cryopreserved. Then spermatozoa were analyzed using the Comet assay, as well as combining this method with digestion with two endonucleases from Escherichia coli (Endonuclease III, that cut in oxidized cytosines, and FPG, cutting in oxidized guanosines). Both storage procedures yielded DNA fragmentation, but only short-term storage oxidative events were clearly detected, showing that oxidative processes affect guanosines rather than cytosines. Cryopreservation increases DNA fragmentation but the presence of oxidized bases was not noticed, suggesting that mechanisms other than oxidative stress could be involved in DNA fragmentation promoted by freezing. [Copyright &y& Elsevier]

Abstract: The first successful artificial insemination (AI) in a rhinoceros was reported in 2007 using fresh semen. Following that success, we decided to evaluate the possibility of using frozen-thawed semen for artificial insemination. Semen, collected from a 35–36 year old Southern white rhinoceros (Ceratotherium simum simum) in the UK was frozen using the directional freezing technique. This frozen semen was used in two intrauterine AI attempts on a 30 years old female rhinoceros in Hungary. The first attempt, conducted 30 days postpartum with an insemination dose of ∼135×106 motile cells, failed. The second attempt, conducted two estrus cycles later with an insemination dose of ∼500×106 motile cells, resulted in pregnancy and the birth of a healthy offspring. This represents the first successful AI using frozen-thawed semen in a rhinoceros, putting it among very few wildlife species in which AI with frozen-thawed semen resulted in a live birth. The incorporation of AI with frozen-thawed semen into the assisted reproduction toolbox opens the way to preserve and transport semen between distant individuals in captivity or between wild and captive populations, without the need to transport stressed or potentially disease carrying animals. In addition, cryopreserved spermatozoa, in combination with AI, are useful methods to extend the reproductive lifespan of individuals beyond their biological lifespan and an important tool for managing genetic diversity in these endangered mammals. [Copyright &y& Elsevier]

Abstract: Approximately 503 of the known species of birds are classified as ‘endangered’ or ‘critical’. Captive propagation programs have proven useful in maintaining genetic diversity and restoring wild populations of certain species, including the Peregrine falcon, California condor and Whooping crane. Artificial insemination (AI) has the potential of solving problems inherent to reproductive management of small, closed populations of endangered birds, including dealing with demographic instability, physical and behavioral disabilities, sexual incompatibility, lack of synchrony, and need to maintain gene diversity. In this review, we address the necessary methods and factors that allow AI to be applied effectively to manage rare bird populations. It is clear that semen availability and quality are the greatest limiting factors to implementing consistently successful AI for birds. Behavioral sensitivity to animal handling and the ability to minimize stress in individual birds also are keys to success. Multiple, deep vaginal inseminations can improve fertility, particularly when semen quality is marginal. Laparoscopic methods of semen transfer also have produced fertile eggs. All of these practices leading to successful AI remain dependent on having adequate basic knowledge on female reproductive status, copulatory behavior, endocrine profiles and duration of fertility, especially as related to oviposition. The overall greatest challenge and highest priority is defining these normative traits, which are highly species-specific. [Copyright &y& Elsevier]

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

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

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

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

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

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

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