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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]

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]

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]

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]

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]

During the fall 2008 breeding season in Louisiana, 2 synchronization protocols for fixed-timed artificial insemination (FTAI) in White-tailed deer were assessed. The objective was to determine if GnRH at FTAI improved pregnancy rates in White-tailed deer. White-tailed does (n= 35) with a mean body weight of 65.8 kg (range: 54.0 to 79.8 kg) and average age of 5.9 years (range: 1.5 to 9.5 years) were stratified by weight, age, and last fawning date into 2 groups. Treatment and control synchronization protocols were randomly assigned to each group. Does were synchronized with a CIDR-G device for 14 d and were then subjected to either FTAI 60 hpost-CIDR removal (control, n= 17) or 100 μg (i.m) injection of GnRH at FTAI 60 h post-CIDR removal (treated, n= 18). At insemination (AI), insemination pipette placement score (IP; 0 = at cervical os to 3 = within uterine body), mucous scores (clear/cloudy, viscous/nonviscous, or no secretions), vulva assessments (hyperemic/nonhyperemic and swollen/not swollen), and sperm progressive motility were recorded. Does were inseminated with frozen-thawed sperm (5 × 107progressively motile cells pre-freeze) from 2 fertile bucks stratified across each treatment group. Starting 28 d following AI, intact bucks (ITB) were introduced into both groups for natural mating. Pregnancy was determined via transrectal ultrasonography 73 or 80 d postinsemination. Does, confirmed pregnant via ultrasonography, had fawns within the reported gestation range of 187 to 222 days. Placentomes were visualized and measured in AI pregnancies (range: 32.7 to 56.2 mm in length), whereas pregnancies derived from ITB presented no identifiable placentomes. However, crown-rump measurements were obtainable from ITB pregnancies (range: 13.4 to 21.7 mm). Five does were not included in the final analyses as they were either lost to predators or removed because of illness. IP (1.4 ± 0.24 v.1.3 ± 0.30; P= 0.671), mucous classifications (3.0 ± 0.35 v.2.5 ± 0.39; P= 0.311), vulva assessments (2.1 ± 0.29, 1.7 ± 0.23; P= 0.223), and sperm motility (1.6 ± 0.16, 1.7 ± 0.22; P= 0.829) were not different for pregnant and nonpregnant AI does, respectively. Treatment did not affect AI pregnancy rates (53 v.27%) or fecundity rates (1.6 v.1.3 offspring/doe) for the GnRH treated and control groups, respectively. Addition of GnRH to a 14-day estrus synchronization protocol did not result in significantly higher pregnancy rates compared with controls. More studies are needed to determine the effect of GnRH on White-tailed pregnancies following FTAI protocols. We have demonstrated that differentiating pregnancies derived from AI and ITB could be accomplished by utilizing transrectal ultrasonography as early as 73 d postinsemination in White-tailed does. [ABSTRACT FROM AUTHOR]

Artificial insemination (AI) involves the accurate detection of oestrus, the ability to pharmacologically induce ovulation, or both. This is particularly important in flying-foxes (Pteropusspp.) that do not have an overt behavioural oestrus. Pregnant mare serum gonadotropin (PMSG) stimulates folliculogenesis and ovulation in the Little Red Flying-fox (P. scapulatus; Towers PA and Martin L 1985 Proc. Aust. Soc. Reprod. Biol. 17, 115). In this study, a dose rate of 15 IU PMSG was used. Our study investigated if 15 IU PMSG would sufficiently induce ovulation in the larger, Black Flying-fox (P. alecto) or if a higher dose (30 IU) would be necessary. Before the mating season, a single injection of PMSG (Folligon®, Intervet Australia Pty Ltd, Bendigo East, Australia) was administered i.m. to eight adult females at 15 IU (n= 4) or 30 IU (n= 4) on Day 0. On Day 4, semen was collected by electro-ejaculation from adult males and inseminated intravaginally into females under isoflurane (Forthane®, Abbott Australasia Pty Ltd, Sydney, Australia) anaesthesia. Ovaries and reproductive tracts were surgically removed on Day 6, fixed, serially sectioned and stained using Gomori''s Stain. Histological sections were examined for evidence of ovarian activity and the presence of ova and spermatozoa in the reproductive tracts. Preliminary observations showed evidence of ovulation in both groups in the form of at least one CL in either ovary and an ovum in the ipsilateral oviduct or uterine horn. Ovaries of females stimulated with 30 IU PMSG differed noticeably from those treated with 15 IU in containing multiple, large, collapsed luteinized follicles with retained oocytes. Vascularization and glandular hypertrophy of the endometrium was also more evident in the higher dose group. No spermatozoa were observed in any of the excised tracts. These results suggest that whereas both doses of PMSG induce ovulation, the administration of 30 IU PMSG may over-stimulate the ovaries. This, in turn, could lead to an unphysiological environment for successful fertilization and embryonic development. A dose level of 15 IU or slightly above may be sufficient for subsequent attempts to stimulate folliculogenesis and ovulation in P. alecto. The absence of spermatozoa suggests that the site of insemination, the number of spermatozoa inseminated, or both requires further investigation if AI is to be successfully implemented in these species. We conclude that the ovarian responses to PMSG indicate that pharmacological induction of ovulation can be successfully achieved and thereby utilized in AI programs of endangered Pteropusspecies. [ABSTRACT FROM AUTHOR]

Two synchronization protocols for fixed-timed AI (FTAI) in White-tail deer were evaluated over a 2-year period. InYear 1, White-tail does (n = 38), with a mean body weight of 55 kg and mean age of 2.4 years, were stratified by weight, age, and last fawning date randomly across 2 estrous cycle synchronization treatment groups. Does received either a CIDR-G device for 14 days (CIDR 14) and underwent AI 60 h post-CIDR removal, or received a CIDR-G device for 7 days (CIDR 7) with 1 mg of estradiol benzoate (i.m.) at CIDR insertion (Day 0), 25 mg of prostaglandin F2α (PGF2α; Dinoprost) on Day 6 and 1 mg of estradiol benzoate (i.m.) on Day 7 with AI occurring 52 h post-CIDR removal. In Year 2, White-tail does (n = 36), with a mean body weight of 60 kg and mean age of 3.9 years, were stratified as described for Year 1 and allotted to 2 treatments. Does received a CIDR-G device for 14 days and were inseminated 60 h post-CIDR removal (Treatment A) or were synchronized in the same way as does inTreatment A but also received 200 IU (i.m.) of eCG (Sigma, St. Louis, MO) at CIDR removal (Treatment B). All does received 1 mg of Domosedan (i.v.) before initiating the AI procedure. In both years, electroejaculated semen was collected from a single buck and frozen for AI. In both years, clean-up bucks were introduced into the pen with does for natural cover at no less than 14 days following AI. A gestation length of 195 7 days was used to determine whether the fawn(s) resulted from AI or from natural mating. In Year 1, FTAI pregnancy rates were not different between the CIDR 14 (56%) and CIDR 7 (24%) treatment groups. Likewise, pregnancy rates were not different between CIDR 14 FTAI (56%) and clean-up bucks (75%). However, the FTAI pregnancy rate was lower (P < 0.001) for the CIDR 7 treatment group (24%) compared with the clean-up bucks (100%). Treatments did not affect fecundity rates; however, those females pregnant from FTAI had lower (P < 0.001) fecundity rates compared with those females pregnant from natural cover (1.2 v. 1.9 fawns/doe). In Year 2, treatment did not affect FTAI pregnancy rates across treatments (33% for Treatment A and 55% Treatment B); however, fewer (P = 0.024) does in Treatment A were pregnant following FTAI (33%) compared with those pregnant from clean-up bucks (75%). There was no difference in FTAI fecundity rates across treatment groups (1.3 v. 1.7 for Treatments A and B, respectively) or between FTAI treatments and natural cover (1.3, 1.7, and 1.5 for Treatments A, B, and clean-up bucks, respectively). These results indicate that the use of a 14-day CIDR synchronization protocol with FTAI occurring 60 h after CIDR removal yields acceptable pregnancy rates of White-tail does following transcervical AI. [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]