The importance and potential of artificial insemination in CANDES (companion animals, non-domestic, endangered species)

Artificial insemination (AI) is the least invasive assisted reproductive technology, and is therefore of great interest to breeders of companion animals, non-domestic, and endangered species (CANDES). This most fundamental artificial breeding technique circumvents physical or behavioral impediments to natural mating and provides the means for genetic exchange between populations without transfer of live animals. In addition, because oocytes grow, mature and are fertilized in vivo and embryos are not subjected to in vitro culture conditions, AI eliminates the epigenetic effects on the female gamete that are inherent in more invasive assisted reproductive technologies. Although the management of CANDES differs significantly from current livestock husbandry practices, the cattle industry is a powerful example of the potential for AI to enhance the genetic health and sustainability of animal populations. Ultimately, successful AI requires sperm of adequate quality and quantity, oocytes that have attained nuclear maturation and cytoplasmic competence, operational gamete transport systems, accurate timing, and proper placement of sperm in the female reproductive tract. Increased understanding of semen collection, evaluation and preservation techniques, estrus synchronization and superovulation, estrus and ovulation detection, and insemination instrumentation is needed for each CANDES before AI success rates will approach those of the livestock industry. Concentrated, collaborative research in these areas must be encouraged among private breeders, universities and zoological institutions to realize the full potential of AI in the management of CANDES.     

Artificial insemination of captive European brown hares (Lepus europaeus PALLAS, 1778) with fresh and cryopreserved semen derived from free-ranging males

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 1 mL 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 22 MHz) 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.     

Additional value of computer assisted semen analysis (CASA) compared to conventional motility assessments in pig artificial insemination

In order to obtain a more standardised semen motility evaluation, Varkens KI Nederland has introduced a computer assisted semen analysis (CASA) system in all their pig AI laboratories. The repeatability of CASA was enhanced by standardising for: 1) an optimal sample temperature (39 °C); 2) an optimal dilution factor; 3) optimal mixing of semen and dilution buffer by using mechanical mixing; 4) the slide chamber depth, and together with the previous points; 5) the optimal training of technicians working with the CASA system; and 6) the use of a standard operating procedure (SOP). Once laboratory technicians were trained in using this SOP, they achieved a coefficient of variation of < 5% which was superior to the variation found when the SOP was not strictly used. Microscopic semen motility assessments by eye were subjective and not comparable to the data obtained by standardised CASA. CASA results are preferable as accurate continuous motility dates are generated rather than discrimination motility percentage increments of 10% motility as with motility estimation by laboratory technicians. The higher variability of sperm motility found with CASA and the continuous motility values allow better analysis of the relationship between semen motility characteristics and fertilising capacity. The benefits of standardised CASA for AI is discussed both with respect to estimate the correct dilution factor of the ejaculate for the production of artificial insemination (AI) doses (critical for reducing the number of sperm per AI doses) and thus to get more reliable fertility data from these AI doses in return.     

Sexual behavior and ejaculate characteristics in Pêga donkeys (Equus asinus) mounting estrous horse mares (Equus caballus)

The objectives were to (i) characterize sexual behavior of donkey stallions (jacks; Equus asinus) during on-farm semen collection using estrous horse mares (mares; Equus caballus); (ii) compare behavior of young (less experienced) versus older (more experienced) jacks; (iii) determine whether semen suitable for artificial insemination (AI) could be collected using mares; and (iv) determine the suitability of using mares in field collection of semen from jacks. Six Pêga jacks (3.5 to 16 yr old), previously conditioned to breed mares, were used. Mount mares were confirmed in estrus by a teaser horse stallion (stallion) and a jack. Semen was collected with an artificial vagina, at intervals of 48 to 72 h (180 collections). The mean ± SD (young [3.5 yr] vs. old [14 to 16 yr]) were Flehmen response frequency, 7.4 ± 5.8 (8.1 ± 3.0 vs. 7.0 ± 2.0); number of mounts without erection, 1.1 ± 1.3 (2.1 ± 1.4 vs. 1.2 ± 0.4, P < 0.05); latency from first exposure to mare to full erection on the ejaculatory mount, 18.3 ± 17.7 min (25.3 ± 21.3 vs. 12.2 ± 6.2, P < 0.05); latency from erection to insertion, 5.1 ± 3.5 sec (5.3 ± 3.8 vs. 4.8 ± 3.2); and duration of copulation from insertion to dismount after ejaculation, 25.4 ± 7.8 sec (22.1 ± 2.9 vs. 28.1 ± 9.3). In all jacks, sexual behavior was generally normal, with the notable absence of open mouth behavior. Mare estrous behavior was markedly less intense than that in the presence of a stallion and usually absent. Semen characteristics were gel free volume, 47.3 ± 28.7 mL; gel volume, 71.8 ± 54.8 mL; total motility, 84.3 ± 6.0%; progressive motility, 74.3 ± 74.5%; sperm vigor, 3.9 ± 0.5 (scale 1 to 5); sperm concentration, 253 × 10⁶ cells/mL; and total number of sperm, 10.3 × 10⁹ cells. Copulation duration was significantly correlated with gel free volume (r = 0.9) and gel volume (r = 0.7). We concluded that (i) the sexual behavior of jacks during semen collection using mares was similar to that reported for natural mating to jennies, (ii) precopulatory and copulatory behavior for the young (less experienced) jacks and older (more experienced) jacks were generally similar (except number of mounts without erection and latency to full erection); (iii) semen obtained using mares as stimulus and mount females was similar to that reported with estrous jennies; and (iv) semen collection from previously conditioned jacks, using estrous mares, was appropriate for field collection of semen.     

Comparison of different osmolalities and egg-yolk composition in processing media for the cryopreservation of red wolf (Canis rufus) sperm

Successful cryopreservation of sperm and the maintenance of a sperm-based genome resource bank have been identified as priorities for the recovery of the endangered red wolf (Canis rufus). The objectives were to improve sperm processing and to determine the relative timing of damage to red wolf sperm during freezing and thawing. Fresh ejaculates (n=37) from adult red wolves (n=15, aged 2-13 y) were collected via electroejaculation and subjected to cooling, freezing and thawing in four TRIS-egg-yolk extender treatments varying in osmolality ( approximately 305 mOsm versus approximately 350 mOsm) and egg-yolk composition (0.8 microm-filtered versus unfiltered). Ejaculates were evaluated for sperm percentage motility, forward progressive motion, and morphological characteristics immediately upon collection and following extension, cooling (prior to freezing) and thawing. Although no single treatment consistently produced superior results, sperm suspended in approximately 305 mOsm extenders exhibited slight losses in motility post-thawing (13 and 7%). Also, sperm suspended in approximately 350 mOsm extenders tended to have slower rates of decline in motility in vitro post-thawing than those stored in approximately 305 mOsm extenders (P=0.55). Finally, extenders incorporating unfiltered egg yolk exhibited a slightly larger ratio of absent to partial acrosomes than did sperm frozen in extenders prepared with clarified egg yolk. For approximately 350 mOsm extenders, most motility loss occurred during the cooling rather than freezing and thawing. In conclusion, these data contribute to knowledge regarding cryopreservation of red wolf sperm.     

Collection and characterization of semen in Mithun (Bos frontalis) bulls

The objective of this study was to collect semen from semiwild Mithun (Bos frontalis) bulls using an artificial vagina (AV) and to determine semen characteristics. Collection of semen with an AV was attempted in five Mithun bulls using both anestrous and estrous Mithun females. No Mithun bull mounted an anestrous female Mithun during 60 trials, but satisfactory mounting, including extension of the penis, occurred in 25 trials with estrous Mithun females. In 15 of these trials, semen was successfully collected in an AV with an internal temperature of 42 to 46 °C. However, in 10 trials with an AV with an internal temperature of 36 to 40 °C, semen was not collected. Mean (± SEM) intervals to first mount and to ejaculation in the AV were 27.9 ± 3.6 sec and 113.8 ± 6.6 sec, respectively. Semen volume and pH were 3.1 ± 0.35 mL and 6.59 ± 0.04, and mean mass activity (scale, 0 to 4), initial sperm motility, live sperm count, sperm concentration, total number of sperm in the ejaculate, and overall sperm length were 2.2 ± 0.3, 78.6 ± 2.6%, 80.7 ± 2.2%, 710.8 ± 66.8 × 10⁶/mL, 2114 ± 364.4 sperm, and 67.9 ± 0.6 μm, respectively. The proportion of morphologically normal sperm was 80.6 ± 0.2%, whereas the proportion with a morphologically abnormal head, midpiece, tail, and acrosome were 4.2 ± 0.4%, 1.6 ± 0.5%, 6.1 ± 1.1%, and 7.1 ± 0.9%, respectively. The mean incidence of tail-less heads and proximal and distal protoplasmic droplets were 0.5 ± 0.1%, 0.3 ± 0.2%, and 2.4 ± 0.3%, respectively. In conclusion, we successfully collected semen from semiwild Mithun bulls with an AV maintained at 42 to 46 °C, and overall, the semen was within the normal range of that collected from fertile domestic bulls.     

Adaptation of ubiquitin-PNA based sperm quality assay for semen evaluation by a conventional flow cytometer and a dedicated platform for flow cytometric semen analysis

The purpose of semen quality evaluation is to predict the fertility potential of the sample in an objective, rapid and inexpensive manner. However, utilization of sperm quality biomarkers such as ubiquitin and lectin Arachis hypogaea agglutinin (PNA) for flow cytometric semen evaluation might eliminate the need for visual assessment by microscopy. Herein, we demonstrate a robust ubiquitin and PNA-based semen evaluation conducted on a simple, easy to operate, dedicated sperm flow cytometer, EasyCyte Plus (IMV Technologies, L'Aigle, France). Semen samples were collected periodically from two dairy bulls, which were subjected to temporary scrotal insults to induce variable semen quality. Samples were labeled with fluorescently-conjugated anti-ubiquitin antibodies (bind exclusively to the surface of defective sperm) and lectin PNA (binds to acrosomal surface in prematurely capacitated and acrosome-damaged sperm). Fluorescent properties of the samples were measured with a conventional flow cytometer (Becton Dickinson FACScan; Becton Dickinson Corp., Franklin Lakes, NJ, USA) and by the EasyCyte (IMV Technologies) instrument. Data from the two flow cytometers were positively correlated for the percentage of PNA-positive sperm with a damaged acrosome (r = 0.47; P < 0.001) and the percentage of ubiquitin-positive, defective sperm (r = 0.68; P < 0.001). Relative intensities of ubiquitin-induced fluorescence in cells with high ubiquitin levels were also positively correlated (r = 0.90). The proportion of sperm with abnormal morphology was positively correlated with ubiquitin-induced fluorescence measured by EasyCyte (IMV Technologies) (r = 0.63; P < 0.001). These observations provided a rationale for the adaptation of a dual ubiquitin-PNA sperm quality assay for flow cytometric semen evaluation.     

Butylated hydroxytoluene inclusion in semen extender improves the post-thawed semen quality of Nili-Ravi buffalo (Bubalus bubalis)

The study was carried out to evaluate the potential impact of butylated hydroxytoluene (BHT) on the frozen-thawed semen quality of Nili-Ravi buffalo bulls. Ejaculated bull semen was extended in a Tris-citrate egg yolk extender containing various concentrations of BHT (0.5, 1.0, 2.0 and 3.0 mM). Semen was frozen at −196 °C using 50 × 10⁶ spermatozoa per 0.5 mL straws. Five straws from each treatment were thawed to assess the semen quality in terms of sperm motility, viability, plasma membrane integrity and acrosomal integrity. Post-thawed sperm motility was determined using a phase-contrast microscope. Viability, plasma membrane integrity and acrosomal integrity were evaluated by the supravital staining, hypo-osmotic swelling test and normal acrosomal reaction, respectively. The highest (P < 0.05) motility, acrosomal integrity and hypo-osmotic swelling response of spermatozoa was achieved by addition of 1.0 and 2.0 mM BHT to semen extender. However, highest (P < 0.05) viability of spermatozoa was achieved by inclusion of 2.0 mM BHT. The higher concentration of BHT (3.0 mM) reduced the motility, acrosomal integrity, viability and hypo-osmotic swelling response of the spermatozoa compared to other concentration used. In conclusion, BHT when added in the semen extender can improve the semen quality of buffalo bulls.     

Differences in semen freezability and intracellular ATP content between the rooster (Gallus gallus domesticus) and the Barbary partridge (Alectoris barbara)

This study aimed to compare viability, ATP content, and DNA integrity of rooster (Gallus gallus domesticus) and Barbary partridge (Alectoris barbara) fresh and frozen spermatozoa in order to identify factors possibly related to differences in semen freezability. Ejaculates were obtained from March to May by the abdominal massage method from 3 adult roosters and 12 adult Barbary partridges. Semen was frozen with different cryoprotectants using Lake's diluents as a base medium: 1) glycerol 11%; 2) glycerol 11% and trehalose 70 mmol/L; 3) dimethylacetamide (DMA) 6%; 4) DMA 6% and trehalose 70 mmol/L. Both fresh and frozen semen showed a lower viability and higher intracellular ATP concentrations in the Barbary partridge compared with the rooster (P < 0.05). In the Barbary partridge, semen viability after thawing did not differ among the 4 media used, but glycerol showed positive effects in avoiding a significant loss of ATP after thawing, compared with DMA containing media (P < 0.05). On the other hand, in the rooster a higher viability was recorded when semen was frozen in glycerol containing media compared to DMA (P < 0.0001), while ATP values significantly decreased after thawing (P < 0.05) without showing any differences among the semen frozen in the 4 different media. DNA integrity, as evaluated by the comet assay, was assessed only in frozen semen. In the Barbary partridge, mean scored parameter did not differ significantly among semen frozen in the 4 different media. In the rooster DNA fragmentation was higher in DMA ctr medium compared with the other media and with values found in Barbary partridge semen frozen in the same medium (P < 0.001). In both species, the addition of trehalose did not show any positive effects on viability, ATP levels and DNA integrity after thawing.In conclusion, species-related differences in semen features exist between the rooster and the Barbary partridge and the wide variation observed in ATP levels may account for differences in semen freezabililty between the two species.     

Controlling contagious agalactia in artificial insemination centers for goats and detection of Mycoplasma mycoides subspecies capri in semen

Many goat artificial insemination (AI) centers in Spain have adopted new measures to control contagious agalactia (CA). To avoid the introduction of male goats carrying mycoplasma organisms subclinically in their external ear canal (auricular carriers) in these centers, two ear swabs and a blood sample are obtained from all candidate animals for polymerase chain reaction (PCR), culture (swabs) and serologic tests to detect the presence of mycoplasmas. In addition, the semen produced at these centers is routinely cultured and PCR tested also to detect the presence of mycoplasmas. One y after the introduction of this program, we tested 48 ear swabs and 24 blood samples from 24 candidates for admission to these AI Centers. Three of these ear swab samples (3/48, 6.25%) scored positive for the presence of mycoplasmas; Mycoplasma agalactiae (Ma) was detected in two samples and Mycoplasma mycoides subsp. capri (Mmc) in one. All animals were serologically negative for Ma. Also, out of 173 semen samples obtained from 137 admitted animals (2 and 3 samples were obtained in 16 and 10 bucks, respectively), one (1/173, 0.56%) was positive for Mmc. Our findings suggest that ear swab and semen samples are useful tools to control CA at AI Centers. The introduction of this program has also resulted in the first detection of Mmc in semen from a naturally infected goat, confirming the ability of this mycoplasma to colonize the reproductive tract of male goats. These results highlight the need to improve control measures in semen producing centers to minimize the risk of CA transmission.     

Favoring the birth of female puppies after artificial insemination using chilled semen diluted with powdered coconut water (ACP-106c)

The objective was to determine the effect of powdered coconut water extender (ACP-106c) on the proportion of female puppies born. Twenty French Bulldog bitches were subjected to natural mating (NM) and, during the subsequent two estrus periods, were bred by intravaginal artificial insemination (AI), using chilled semen (from the same males) diluted in Tris-egg yolk (AI-Tris) or ACP-106c (AI-ACP-106c). Fresh semen was cooled to 5 °C and maintained at that temperature for 6 h, rewarmed (37 °C for 30 s), and used for AI. Pregnancy and whelping rates following NM were both 100% and were both 90.0% following AI with either extender. Litter size (mean ± SD) was 5.4 ±1.1, 4.7 ± 2.0, and 5.1 ± 2.0 (P > 0.05) for NM, AI-Tris, and AI-ACP-106c, respectively. Furthermore, for these groups, the number of female vs. male puppies born were 2.6 ± 0.6 vs. 2.8 ± 1.0, 2.2 ± 1.0 vs. 2.5 ± 1.1, and 3.4 ± 1.6 vs. 1.8 ± 1.2 (P < 0.05 for AI-ACP-106c only). In conclusion, our hypothesis was supported; AI of semen in ACP-106c extender resulted in a significantly higher proportion of female puppies. Furthermore, this extender yielded acceptable litter size and rates of pregnancy and whelping.     

Cryopreservation of collared peccary (Pecari tajacu) semen using different freezing curves,straw sizes,and thawing rates

The objective of this study was to verify the effect of different freezing curves, straw sizes, and thawing rates on the cryopreservation of collared peccary semen. Twelve ejaculates were obtained from captive adult males by electroejaculation, and evaluated for sperm motility, kinetic rating, viability, morphology, and functional membrane integrity. The ejaculates were diluted in a coconut water extender (ACP-116c) with egg yolk and glycerol, packaged into 0.25 mL or 0.50 mL plastic straws and cryopreserved in liquid nitrogen following a slow (−10 °C/min) or a fast (−40 °C/min) freezing curve. After one week, samples were thawed at 37 °C/1 min or 70 °C/8 s and evaluated as reported for fresh semen, and also for kinematic parameters (computerized analysis). A significant decrease in sperm motility and kinetic rating was observed after glycerol addition at 5 °C and also after thawing for all the treatments (P < 0.05). Regarding post-thaw semen variables, no differences were verified between freezing curves when the same straw size and thawing rate were taken as reference (P > 0.05). In general, values for sperm characteristics found after thawing at 37 °C were better preserved than at 70 °C (P < 0.05), both in the use of 0.25 mL or 0.50 mL straws, which were similar for semen packaging (P > 0.05). The evaluation of the kinematic parameters of sperm motility confirmed these results at values varying from 20% to 30% motile sperm for the samples thawed at 37 °C, and values fewer than 12% motile sperm for samples thawed at 70 °C (P < 0.05). In conclusion, we recommend the use of a fast freezing curve that reduces the time spent on the cryopreservation of collared peccary semen, which could be packaged both in 0.25 mL or 0.50 mL straws, but the thawing should be conducted at 37 °C/1 min.     

Comparison of electrostimulation methods for semen recovery in the rhesus monkey (Macaca mulatta)

Electroejaculation of 17 rhesus monkeys was performed at intervals during a 15-month period using both penile and rectal probe stimulation. The semen quality was compared for the two stimulation methods. Both methods were effective in approximately 90% of attempts, and there was no difference in fertilizing capacity of the sperm. A seasonal difference in semen quality was detected, and samples recovered by penile stimulation showed higher sperm count.     

Use of two anesthetic combinations for semen collection by electroejaculation from captive coatis (Nasua nasua)

The objective was to compare the effects of ketamine-xylazine or tiletamine-zolazepam combinations as anesthetic protocols for captive coatis (Nasua nasua) for semen collection by electroejaculation. Five mature male coatis were physically restrained and then anesthetized by im injections of ketamine (10 mg/kg) plus xylazine (1 mg/kg) or a tiletamine-zolazepam combination (8 mg/kg). For the two combinations, additional quarter-doses of ketamine or the tiletamine-zolazepam combination were administered when necessary. Semen was collected by electroejaculation and immediately evaluated for color, volume, pH, sperm motility, vigor, morphology, acrosomal integrity, and percentage of live cells. Overall, collection of nine ejaculates was attempted from five animals for each treatment. Regardless of the anesthetic combination, all animals developed an erection during each attempt to collect semen. Ejaculates were obtained in all (9 of 9) attempts that used ketamine-xylazine for anesthesia, but in only 3 of 9 attempts (P < 0.05) when tiletamine-zolazepam was used. All ejaculates contained sperm, with no significant differences in semen characteristics between the two anesthetic combinations. Recovery was smooth in all animals. In conclusion, semen collection by electroejaculation in coatis was significantly more successful with the use of a ketamine-xylazine combination than with tiletamine-zolazepam.     

Improvement of liquid and frozen-thawed semen quality of Nili-Ravi buffalo bulls (Bubalus bubalis) through supplementation of fat

The aim of the study was to investigate the effects of dietary fat on quality of liquid and frozen-thawed semen of Nili-Ravi buffalo bulls. Adult bulls (n = 21) were fed a balanced ration (Con; n = 7) or the same ration either containing sunflower oil (SF-O; n = 7) or whole sunflower seeds (SF-S; n = 7) for 63 days. Body weight and body condition score of each bull was recorded on days 0, 30 and 60 of the experiment. Semen was collected on days 39, 46, 53 and 60, frozen by a fast method and stored at −196 °C for 24 h. Sperm motility was assessed using a bright field microscope. Plasma membrane integrity of fresh and frozen-thawed spermatozoa was assessed using a hypo-osmotic swelling (HOS) assay. The concentration of spermatozoa and volume of semen was not different among groups on various days of collection. Sunflower-enriched diets did not affect the motility and number of HOS-positive spermatozoa in the fresh semen. Motility and HOS of post-thawed spermatozoa were higher (p < 0.05) in bulls fed the sunflower-enriched diets. Similarly, diets did not affect the body condition score and body weight of bulls. In conclusion, feeding of sunflower oil or sunflower seed as fat sources can improve the quality of buffalo bull spermatozoa.     

Effect of semen collection method on sperm motility of gray wolves (Canis lupus) and domestic dogs (C. l. familiaris)

Genetic management of Mexican gray wolves includes semen banking, but due to the small number of animals in the population and handling restrictions, improvements in semen collection and cryopreservation rely on results from studies of domestic dogs. Semen collection from wolves requires anesthesia and electroejaculation, which introduce potentially important variables into species comparisons, as dog semen is typically collected manually from conscious animals. To investigate possible effects of collection method on semen quality, we compared semen collection by the traditional manual method and by electroejaculation (EE) in a group of dogs (n = 5) to collection by EE only in wolves (n = 7). Samples were divided into two aliquots: neat or diluted in Tris/egg yolk extender, with motility evaluated at intervals up to 24 h. There were no differences (P > 0.10) in sperm motility in either neat or extended samples at 24 h from EE dogs and wolves, although motility of the wolf neat samples declined more rapidly (P < 0.05). However, there were differences (P < 0.01) between EE and manually collected dog semen in motility at 24 h, in both the neat and extended samples. Therefore, general motility patterns of dog and wolf semen collected by EE were similar, especially when diluted with a Tris/egg yolk extender, but sperm collected from dogs by EE did not maintain motility as long as manually collected samples, perhaps related to the longer exposure of EE samples to more prostate fluid.     

Successful ovulation induction and laparoscopic intrauterine artificial insemination in the clouded leopard (Neofelis nebulosa)

Exogenous gonadotropins and a laparoscopic intrauterine artificial insemination (AI) technique were assessed for effectiveness in the clouded leopard (Neofelis nebulosa), a species difficult to breed in captivity due to severe mate incompatibility. Fourteen hormone trials using 10 female clouded leopards were performed to evaluate the ability of 50, 100, or 200 i.u. pregnant mares' serum gonadotropin (PMSG) and 75 or 100 i.u. human chorionic gonadotropin (hCG) to induce folliculogenesis and ovulation, respectively. Laparoscopic evaluation of ovarian activity was conducted at 29–48 hr after hCG administration. Time of ovulation in PMSG/hCG-treated clouded leopards was approximately 38–39 hr after hCG. Excessive follicular development was observed using the high hormone dosages (200 i.u. PMSG/100 i.u. hCG), whereas the lower dosages avoided ovarian hyperstimulation. Previous ovulation sites and mature corpora lutea were detected upon laparoscopic examinations in two of the 10 females housed alone, indicating that this species occasionally spontaneously ovulates. Five females were inseminated by depositing electroejaculated, washed sperm transabdominally into the proximal aspect of each uterine horn. One postovulatory female, previously treated with 100 i.u. PMSG and 75 i.u. hCG and inseminated in utero with 88 × 10⁶ motile sperm at 45 hr post-hCG, produced a pregnancy and two live cubs after an 89 day gestation. These results demonstrate: (1) an exquisite ovarian sensitivity to exogenous gonadotropins in clouded leopards; and (2) that artificial insemination has the potential of resulting in offspring in this species. (This article is a US Government work and, as such, is in the public domain in the United States of America.) © 1996 Wiley-Liss, Inc.     

Estrous synchronization in the gaur (Bos gaurus): Behavior and fertility to artificial insemination after prostaglandin treatment

The gaur (Bos guarus) is an endangered species that is a wild ancestor of domestic cattle. This study was conducted to evaluate 1) the efficacy of prostaglandin F_2α (PG) to synchronize estrus in gaur, 2) behavior of male and female gaur around estrus, and 3) fertility after artificial insemination (AI) at the PG-induced estrus. Six female gaur were utilized, along with a vasectomized gaur bull used to aid in detecting estrus. All females were given two i.m. injections of PG (25 mg/injection) 11 days apart, and monitored for estrus for 120 hr (48 hr via chinball marks, followed by 72 hr continuous observation) after the second PG injection. Three of the six females were in estrus during the 120-hr evaluation period. One female was in estrus prior to 48 hr after the second PG injection, and two females were observed in estrus during the 72-hr continuous observation period. When a female was observed in estrus (standing to be mounted by the vasectomized bull), she was bred by AI at 12 and 24 hr after the onset of estrus. The four females not observed in estrus, including the one marked during the first 48 hr, were bred by AI at 80 and 92 hr after the second PG injection. Of the two gaur females observed in estrus, one female was first mated by the vasectomized bull at 77 hr and the second female was mated at 98 hr after PG. Both females exhibited very short durations of receptivity (less than 4 hr). The second female observed in estrus became pregnant after AI and gave birth to a healthy gaur calf after a 299-day gestation. It appears that female gaur can be synchronized with PG techniques developed for domestic cattle. These data should provide useful information for programs studying and maintaining this endangered species and may have relevance for the cattle industry, since the gaur could provide a source of diverse ancestral genetic material.     

Collection and evaluation of semen from the three-toed sloth (Bradypus tridactylus)

Sloths (Bradypus sp.) are extremely sensitive animals that suffer with the destruction and fragmentation of forests. They present a low population growth rate and need to be further studied for the preservation of the specie. Thus, the aim of this study was to establish an efficient semen collection protocol as well as characterize sperm concentration, motility and morphology in order to contribute with information about the reproductive traits of this specie, which has never been described in the literature before. For that, nine Bradypus tridactylus males were captured during the wet season and six during the dry season, in Manaus (AM), located in the north region of Brazil, semen was collected by electroejaculation with shocks given in sequences of progressive intensities (minimum 20 mA and maximum 60 mA). All animals ejaculated small volumes of semen and in some of them, the volume ejaculated was not enough for a complete spermiogram. Physical characteristics observed on the collections of the wet season were different from those seen in the specimen collected in the dry season. Motility an vigor was very low and did not show forward progression, only oscillatory movement. After Spermac stain, spermatozoa presented a wide variety of defects; however, the differences in morphology were not significant between seasons. The morphology assessed by scanning electron microscopy shows that the head in both groups could be elongated, short or could have a base narrower than the apex and the midpiece narrowed abruptly, forming a nip in its transition to the tail. Although further studies are necessary to verify our preliminary findings concerning seasonal variation in sperm quality, these results demonstrate that semen can be safely collected from sloths by electroejaculation and provide the first reports of semen characteristics in this species.