Improved method for artificial insemination in the great apes

Artificial insemination in the great apes has not achieved its potential as a tool in maintenance of the endangered captive population. Three factors can influence the success rate of artificial insemination: sperm preparation, site of insemination, and timing of insemination. We have tried to optimize methods regarding these three steps. A modified method for insemination is described which has resulted in a 21% success rate (six term pregnancies from 29 inseminations) in the chimpanzee and which has successfully initiated a pregnancy in a gorilla.     

Successful artificial insemination in the giant panda (Ailuropoda melanoleuca) at ueno zoo

Successful breeding of the giant panda (Ailuropoda melanoleuca) following artificial insemination was achieved at the Ueno Zoo in 2 consecutive years (1985 and 1986). The first cub, born in June 1985, unfortunately died 43 hours after birth from being crushed by the mother panda; the second cub, born in June 1986, has been growing in good health. Electroejaculation and artificial insemination procedures were performed after immobilization with diazepam (0.1 mg/kg) and ketamine HCL (4.0–5.0 mg/kg). Semen of the male panda was collected by electroejaculation using a rectal probe with a diameter of 2.0 cm and with eight rings as electrodes. Stimulation of the male was given with 3 V (30–40 mA) over a 5-sec period with 5-sec intervals. The female panda exhibited estrus between late February and early March in 1985 and also between mid-january and early February 1986. Increased excretion of urinary total estrogen showed coincidentally at maximum behavioral estrus, and a gradual rise of pregnanediol level was followed by artificial insemination. The gestational length for the first pregnancy was 110 days and that of the second 121 days.     

Space use selectivity by chimpanzees and gorillas in an indoor-outdoor enclosure

Understanding the relationship between physical environments and nonhuman primate behavior is a key element for effective care and management in a range of settings. The physical features of the captive environment, including not only gross useable space but also environmental complexity, can have a significant influence on primate behavior and ultimately, animal welfare. But despite this connection, there remains relatively little conclusive data on how captive primates, especially great apes, use the spaces provided to them, especially in modern, indoor-outdoor enclosures that have become more prevalent in recent years. In this study, we used four years of detailed data on where 23 great apes (chimpanzees and gorillas) positioned themselves within a modern, indoor-outdoor zoo enclosure to determine not only how the apes utilized their space but also how access to outdoor areas affected their spatial selectivity. We found that both species used relatively little of their available space: chimpanzees and gorillas spent half their time in only 3.2 and 1.5% of their useable three-dimensional space, respectively. Chimpanzees utilized the outdoor space more than gorillas, but access to the outdoors did not affect space selectivity in the indoor area for either species. Although both species of ape were highly selective in their space use, consideration should be given to the importance of providing the choice to locate in a variety of spaces, including outdoor areas. These data represent an extremely detailed account of space selectivity by great apes in an indoor-outdoor environment and have substantial implications for future facility design and captive primate management.     

Problems and experiences in performing artificial insemination in bonobos (Pan paniscus)

A report is given on the problems and the procedure of artificial insemination in the bonobo (Pan paniscus). In addition, the different possibilities and methods of the process of collecting sperm, its conservation, and determining the time of ovulation are discussed, as well as the various insemination techniques and their usage under practical conditions in zoological gardens.     

Birth of a chimpanzee (Pan troglodytes) after artificial insemination with cryopreserved epididymal spermatozoa collected postmortem

A male chimpanzee (Pan troglodytes) suddenly died of acute hemorrhagic enteritis at the age of 18 years. Within 24 hours after its death, a large quantity of spermatozoa of excellent quality was recovered from the distal cauda epididymides and was subsequently cryopreserved. After storage for 67 days, the frozen spermatozoa were thawed and inseminated in an adult, normal cycling, nulliparous female. The optimal day for insemination was estimated by monitoring the swelling of the female’s sex skin and urinary luteinizing hormone concentrations. Pregnancy was confirmed by a urinary chorionic gonadotropin test, and a normal female infant was born after 214 days of gestation. This birth demonstrates that distal cauda epididymal spermatozoa recovered from a dead male can be cryopreserved and successfully inseminated in a female chimpanzee. Zoo Biol 20:135–143, 2001. © 2001 Wiley‐Liss, Inc.     

Transcervical artificial insemination in the domestic ferret (Mustela putorius furo)

The research was undertaken to develop a successful nonsurgical procedure for artificially inseminating ferrets. A fiberoptic endoscope used in conjunction with a specially designed speculum and catheter permitted cervical catheterization and intrauterine insemination. Sperm were collected from the cauda epididymides of 10 discarded breeder males; the number of sperm in diluted samples used for insemination ranged from 4.4–13.6 × 10⁶/100 μl with progressive motility of sperm ranging from 40 to 60%. Sperm collected from each male were diluted with an egg-yolk extender (TEST) and used to inseminate 8–12 females, with deposition of sperm intravaginally or transcervically into the uterine body 0 or 24 hr after an ovulatory injection of human chorionic gonadotropin (hCG). The vaginal inseminations were used as a control, and no pregnancies resulted after insemination of 26 females. Intrauterine inseminations resulted in 4/24 (17%) of the ferrets pregnant when hCG administration was coincident with insemination, and 19/24 (79%) of the ferrets were pregnant when inseminations were done 24 hr after hCG administration. All inseminated females were euthanized on day 20 after insemination to count fetuses. The mean number of fetuses was 3.1 (range, 1–8). The number (millions) of motile sperm inseminated (X) had a significant effect on the percentage of fetuses (Y). Regression analysis indicated a linear relationship between the two variables, with an R² value of 0.99 and a line of best fit described by the equation Y = 0.029 + 0.034 X. This paper is the first report of transcervical artificial insemination in the domestic ferret (Mustela putorius furo). The method can serve as a model for application to ferrets and other mammals, particularly endangered species. Zoo Biol 17:393–404, 1998. © 1998 Wiley-Liss, Inc.     

Successful artificial insemination in the corn snake,Elaphe gutatta,using fresh and cooled semen

The design and implementation of assisted reproductive technology to improve genetic diversity and augment captive populations is an important but rarely applied research field in reptiles. Using the corn snake (Elaphe gutatta) as a model, the Henry Doorly Zoo recently produced offspring born as a result of artificial insemination using both fresh, diluted semen, and diluted semen stored at refrigeration for 3 days. Semen was collected noninvasively from sexually mature male corn snakes using a gentle massaging technique, extended in medium then inseminated into the oviducts of adult females. Using molecular genetic techniques to confirm or refute the success of the insemination using primers developed for the black rat snake, Elaphe obsolete, all possible parents and offspring genotypes were evaluated. A paternity‐by‐exclusion analysis verified that the offspring were in fact a result of artificial insemination. Zoo Biol 26:363–369, 2007. © 2007 Wiley‐Liss, Inc.     

Bovine pregnancy and nonreturn rates following artificial insemination using a covered sheath

Contamination transferred into the uterus from external genitalia during artificial insemination (AI) has been hypothesized to cause lowered bovine pregnancy rates (PR). Using aseptic techniques, there is still a possibility of uterine contamination during routine AI. Two experiments were conducted to evaluate the effect of two types of sheath covers (CS) placed over the conventional French Medium Syringe assembly (FMS) used for AI. Their use entailed passing the assembly to the external os of the cervix, pushing the FMS through the CS and manipulating the FMS to the cervical uterine junction in the normal manner. Fifty-six day non-return rate (NRR) in dairy and actual PR in beef cattle were evaluated. In Experiment 1, 30 professional technicians were employed to inseminate 7, 387 dairy cows, while in Experiment 2, six technicians with varying levels of experience inseminated 416 beef cows. Least-squares means for NRR in dairy cattle were 78% using a CS and 79% without. Means for PR in beef cattle were 57% using a CS and 62% without. In Experiment 2, the overall PR was lower in Trial l than in other trials (P<0.05). Since some technicians improved with time, the difference due to trial was attributed to technician variation in gaining experience with a CS. Results indicate that general use of a CS in routine AI of apparently healthy cows will not increase PR.     

Profitability of a dairy sheep genetic improvement program using artificial insemination

This simulation study investigated the farm-level economic benefits of a genetic improvement scheme using artificial insemination (AI) with fresh ram semen in dairy sheep of the Chios breed in Greece. Data were collected from 67 farms associated with the Chios Sheep Breeders' Cooperative 'Macedonia', describing the percentage of ewes that would be artificially inseminated in the flock, pregnancy rate, annual ram costs that could be saved using AI rather than natural mating, expected improvement in milk production, annual costs of semen and feed, milk price and number of years of AI usage. The study considered 77 760 possible scenarios in a 3 × 4 × 4 × 3 × 3 × 3 × 4 × 15 factorial arrangement. Analysis of variance was used to investigate the effect of each factor on farm profitability. All factors considered were statistically significant (P < 0.001), but their effect varied. The number of years using AI had the greatest effect on profitability and farmers should become aware that using AI is a long-term investment. Semen price, pregnancy rate and improvement in milk production also had substantial effects. The price of milk and feed had a considerably lower effect on profitability, as did the annual cost of maintaining rams that would be replaced by AI. A positive annual and cumulative return was achieved in the model within the first 6 years. The cost of semen was estimated at 8€ to 10€ per dose for the first 5 years. Where the annual improvement in milk production was 1% of annual phenotypic mean (e.g. 3.0 kg) profitability of the scheme was improved greatly.     

Birth of offspring following artificial insemination in the common marmoset,Callithrix jacchus

The objective of the study was to develop a method for artificial insemination (AI) in the common marmoset, a New World primate species. For AI to be successful, sperm must be deposited at an appropriate site and time in the female reproductive tract, details of which are currently not available for Callitrichid species. Epididymal sperm were deposited in the cervix of 18 marmoset monkeys (Callithrix jacchus) around the time of expected ovulation using either 3, 2, or 1 inseminations. Six out of 18 females conceived, resulting in the first reported births following AI in this species. These pregnancies show that the presence of coagulum in the vagina and the stimulus of the female reproductive tract by natural mating are not essential for effective sperm transport in this species. Although 3 different timing regimes for sperm deposition relative to ovulation were employed, no protocol was demonstrably better than the others in terms of number of conceptions. The proportions of motile, live, and morphologically normal sperm in the suspensions used for AI were comparable with published values for ejaculates from fertile male macaques. These preliminary results indicate that births are possible following AI in marmosets: the technique could be used to aid effective genetic management of the species and possibly to facilitate captive breeding of endangered Callitrichids. Am J Primatol 41:37–43, 1997. © 1997 Wiley-Liss, Inc.     

Comparison of artificial insemination and natural service cost effectiveness in dairy cattle

Reproductive efficiency in the dairy herd is the most important factor for its economic success and a major concern for dairy farmers when using artificial insemination (AI) or natural service (NS). Our objectives were to estimate, compare and analyse the costs associated with breeding cattle by do-it-yourself (DIY) AI and NS and identify the factors that influence them, under typical dairy farming conditions in Greece. A simulation study was designed based on data from 120 dairy cattle farms that differed in size (range 40 to 285 cows) and milk production level (4000 to 9300 kg per cow per year). Different scenarios were employed to estimate costs associated directly with AI and NS as well as potentially extended calving intervals (ECI) due to AI. Results showed that bull maintenance costs for NS were €1440 to €1670 per year ($1,820 to $2,111). Direct AI costs were higher than those for NS for farms with more than 30 cows and ECI constituted a considerable additional burden. In fact, amongst the factors that affected the amount of milk needed to cover total extra AI costs, number of days open was the dominant one. Semen, feed and heifer prices had a very small effect. When, hypothetically, use of NS bulls results in a calving interval of 12 months, AI daughters with a calving interval of 13.5 months have to produce about 705 kg of additional milk in order to cover the extra cost. Their actual milk production, however, exceeds this limit by more than 25%. When real calving intervals are considered (13.0 v. 13.7 months for NS and AI, respectively) AI daughters turn out to produce more than twice the additional amount of milk needed. It was concluded that even under less than average management conditions, AI is more profitable than the best NS scenario. The efficient communication of this message should be a primary concern of the AI industry.     

Induction of ovulation and successful artificial insemination in a Persian leopard (Panthera pardus saxicolor)

Techniques of artificial insemination have not been readily applied to zoo animals. The nonsurgical approach has been unsuccessful in nondomestic felids until the present study where pregnancy was achieved in a Persian leopard. The demonstration of this technique should encourage renewed attempts to artificially inseminate nondomestic felids.     

Comparison of prostaglandin- and progesterone-based protocols for timed artificial insemination in sheep

The objective was to compare the reproductive performance of a new PGF_2α-based timed artificial insemination (TAI) protocol in sheep (Synchrovine®: two doses of PGF_2α, 7 d apart) to a traditional progesterone-eCG (P4-eCG) protocol, considering the effects of seminal state, AI-times, and AI-pathway. Three experiments involving 1297 multiparous Australian Merino ewes were done during the physiologic breeding season (location 32 °S-57 °W). Reproductive performance was assessed as non-return rate to service 21 d after AI (NRR21d), based on detection with androgenized wethers, as well as Fertility (pregnant/inseminated ewes), Prolificacy (fetuses/pregnant ewe), and Fecundity (fetuses/inseminated ewe), which were based on transabdominal ultrasonography 50 d after TAI. In Experiment 1, Synchrovine® treated ewes TAI cervically with fresh semen at 42, 48, or 54 h had similar NRR21d (0.51, 0.46, 0.57), Fertility (0.27, 0.31, 0.26), and Fecundity (0.29, 0.32, 0.27), all of which were lower (P < 0.05) than in a control P4-eCG group inseminated at 54 h (0.61, 0.48, 0.52, NRR21d, Fertility and Fecundity respectively). In Experiment 2, using chilled semen and cervical TAI, Synchrovine® treated ewes inseminated at 42 h yielded lower (P < 0.05) NRR21d, Fertility and Fecundity (0.28, 0.06, 0.06) compared to 48 (0.43, 0.24, 0.24) and 54 h (0.44, 0.22, 0.23). In Experiment 3 with chilled semen, Synchrovine® treated ewes TAI into the cervix at 51 or 57 h were similar in NRR21d (0.16 vs 0.20), Fertility (0.12 vs 0.14), and Fecundity (0.12 vs 0.15), respectively; but lower (P < 0.05) than P4-eCG treated ewes TAI at 54 h (0.34, 0.28, and 0.33 for NRR21d, Fertility and Fecundity respectively). Synchrovine® treated ewes intrauterine TAI at 51 or 57 h yielded similar NRR21d (0.51 vs 0.58), Fertility (0.43 vs 0.51), and Fecundity (0.45 vs 0.56) respectively, but lower (P < 0.05) results compared to P4-eCG treated ewes (0.75, 0.71, and 0.88 for NRR21d, Fertility and Fecundity respectively). In conclusion, AI-time in Synchrovine® treated ewes with fresh semen might be extended (42 to 54 h after the second PGF_2α), but should be delayed to 48-54 h with chilled semen and cervical AI. Independent of the seminal state, AI-time or AI-pathway, Synchrovine® yielded lower reproductive results than a conventional P4-eCG protocol.     

Seasonal fluctuations in the response of Italian Mediterranean buffaloes to synchronization of ovulation and timed artificial insemination

A comprehensive study of the efficiency of synchronization of ovulation and timed artificial insemination (TAI) was undertaken in a large group of Italian Mediterranean buffaloes at a commercial dairy. A total of 2791 synchronization protocols were carried out on 857 animals over 3 years. Of these protocols, 823 (29.5%) did not proceed beyond Day 7 (due to the absence of a vascularized CL) and 620 (22.2%) were discontinued on Day 10 (due to the absence of follicles >1.0 cm and tonic uteri); hence, 1443 (51.7%) protocols did not progress to TAI. Data were analyzed for four periods: P1, transition to spring (from breeding season to low breeding season); P2, low breeding season; P3, transition to fall (low breeding season to breeding season); and P4, breeding season. No differences were found among the four periods in terms of the proportion of protocols that did not result in TAI. Of the 857 buffaloes, 660 (77%) conceived and delivered a calf. The average number of TAI per pregnancy was 2.1 and ranged from 1.9 to 2.3 across years. Logistic regression analysis showed that buffaloes that calved during P3 had a higher odds ratio for pregnancy (1.380; P < 0.05) than buffaloes that calved in other periods. Pregnancy was also influenced by the calving to service period (odds ratio = 0.977; P < 0.01) and the pregnancy per AI (P/AI) at successive TAI (odds ratio = 1.480; P < 0.01). The pregnancy per AI at the first TAI (424/857, 49.5%) was greater (P < 0.01) than in subsequent TAI. The occurrence of late embryonic mortality (between Days 27 and 45 after TAI) was similar among the four periods. These findings indicated that there are distinct seasonal differences in the response of Italian Mediterranean buffaloes to synchronization and TAI.     

Environmental and genetic variation factors of artificial insemination success in French dairy sheep

Artificial inseminations (n = 678 168) recorded during 5 years in five French artificial insemination (AI) centres (2 'Lacaune', 1 'Manech tête rousse', 1 'Manech tête noire' and 1 'Basco béarnaise') were analysed to determine environmental and genetic factors affecting the insemination results. Analyses within centre-breed were performed using a linear model, which jointly estimates male and female fertility. This model combined four categories of data: the environmental effects related to the female, those related to the male, the non-sex-specific effects and finally the pedigree data of these males and females. After selection, the environmental female effects considered were age, synchronisation (0/1) on the previous year, total number of synchronisations during the female reproductive life, time interval between previous lambing and insemination, already dry or still lactating (0/1) when inseminated, and milk quantity produced during the previous year expressed as quartiles intra herd * year. The environmental male effects were motility and concentration of the semen. The non-sex-specific effects were the inseminator, the interaction herd * year nested within the inseminator, considered as random effects and the interaction year * season considered as a fixed effect. The main variation factors of AI success were relative to non-sex-specific effects and to female effects. Heritability estimates varied from 0.001 to 0.005 for male fertility and from 0.040 to 0.078 for female fertility. Repeatability estimates varied from 0.007 to 0.015 for male fertility and from 0.104 to 0.136 for female fertility. These parameters indicate that genetic improvement of AI results through a classical polygenic selection would be difficult. Moreover, in spite of the large quantity of variation factors fitted by the joint model, a very large residual variance remained unexplained.     

Bio-economic and operational feasibility of introducing oestrus synchronization and artificial insemination in simulated smallholder sheep breeding programmes

Low selection intensity due to few selection candidates available at any one time due to thinly spread year-round lambings in villages and prohibitively large nucleus requirements to provide sufficient improved rams to the production tier are the major challenges for designing effective village-based and central nucleus-based breeding programmes, respectively, for smallholder sheep farmers. To tackle these challenges, we used deterministic simulation to design three schemes in village-based programmes introducing hormonal oestrus synchronization (natural oestrus (VNE), single oestrus synchronization (VSE1) and double oestrus synchronization (VSE2)) and three schemes in central nucleus programme introducing artificial insemination (AI) (natural mating with nucleus sizes of 5% (CNM1) and 1% (CNM2) of the total ewe population and natural mating in breeding tier and AI in production tier (CAI)). The schemes were evaluated for their bio-economic and operational feasibility, taking Bonga sheep of Ethiopia as a case study. The selection intensities achieved in VNE, VSE1 and VSE2 were 2.0, 2.3 and 2.4, respectively, for selecting rams for the breeding tier and 0.0, 0.8 and 1.0, respectively, for the production tier. The profits per ewe per year from VNE, VSE1 and VSE2 were Birr 12.2, 21.7 and 24.5, but the profit from VNE for the production tier was zero. CAI generated more genetic gains in the breeding objective (Birr 4.8) than CNM1 (Birr 2.5) and CNM2 (Birr 0.0) in the production tier. However, CAI was less profitable than CNM1 and CNM2. In conclusion, hormonal oestrus synchronization was found to be a feasible technological aide to accelerate genetic progress in village-based programmes. CNM1 and CNM2 could not be recommended as CNM1 requires large nucleus of 10 325 ewes and CNM2 results in zero genetic gain in the production tier. CAI could overcome the challenge in central nucleus programmes, namely unaffordable large nucleus, but the scheme needs to be subsidized by the public sector to be economically feasible for farmers.     

Is Doublesynch protocol a new alternative for timed artificial insemination in anestrous dairy cows

This is the very first report that suggests high pregnancy rates can be obtained with use of the Doublesynch protocol in anestrous dairy cows. Recently, a new synchronization method has been developed (Doublesynch) that resulted in synchronized ovulations both after the first and second gonadotropin-releasing hormone (GnRH) treatments. It was suggested that this protocol has the potential to increase the pregnancy rates in primiparous dairy cows. The aim of the current study was to confirm the success of the Doublesynch protocol and further to investigate the effect of this method on pregnancy rates in anestrous cows. Lactating primiparous Holstein (Bos taurus) cows (n = 165) between 60 and 172 d postpartum were monitored twice with 10-d intervals (on Days -10 and 0) by ultrasonography, and blood samples were collected. Cows were classified as anestrous if both blood samples had progesterone (P4) concentration <1 ng/mL and as cyclic if at least one of the two samples had P4 concentration ≥1 ng/mL. Cyclic cows were classified again as cyclic-high P4 (having an active corpus luteum) if the second blood samples had P4 concentrations ≥1 ng/mL and as cyclic-low P4 if P4 concentrations were <1 ng/mL on Day 0. Then, the cows classified as anestrous (n = 51), cyclic-high P4 (n = 63), or cyclic-low P4 (n = 51) were put into two treatment groups (Ovsynch or Doublesynch) randomly to establish six groups. Cows in the Ovsynch group were administered a GnRH (lecirelin 50 μg, im) on Day 0, PGF (Prostaglandin F2 alpha, D-cloprostenol 0.150 mg, im) on Day 7, and a second dose of GnRH 48 h later. Cows in the Doublesynch group were administered a PGF on Day 0, GnRH on Day 2, a second PGF on Day 9, and a second GnRH on Day 11. Timed artificial insemination (TAI) was performed 16 to 20 h after the second GnRH in both treatment groups. Pregnancy diagnosis was conducted (by ultrasonography) 45 ± 5 d after TAI. In anestrous cows and those with high and low progesterone concentration at treatment onset, Doublesynch treatment led to markedly increased pregnancy rates with respect to Ovsynch treatment (P < 0.05). On the overall analysis of data, it was revealed that the Doublesynch method increased pregnancy rates by 43 percentage units (29.8% vs. 72.8%, P < 0.0001) in relation to Ovsynch. Pregnancy rates of cows having small, medium, or large follicles at the day of second GnRH administration were similar in the Doublesynch group (70.4%, 85.2%, and 63.0%, respectively; P > 0.05), whereas pregnancy rates reduced dramatically as follicle size increased in the Ovsynch group, particularly in cows with follicles greater than 16 mm (45.5%, 28.1%, and 5.3%, respectively; P < 0.05). Our results confirm and support observations that the Doublesynch protocol increases the pregnancy rates in postpartum primiparous cows as reported previously. Our data also demonstrate that the Doublesynch method increases the pregnancy rates in anestrous cows. Thus, these data suggest that the Doublesynch protocol can be used to obtain satisfactory pregnancy rates after TAI in both anestrous and cycling primiparous dairy cows regardless of stage of estrous cycle.     

Sperm distribution and fertilization after unilateral and bilateral laparoscopic artificial insemination with frozen-thawed goat semen

Generally, laparoscopic artificial insemination (LAI) provides a higher success rate than of cervical insemination in goats. However, the sperm distribution after LAI in goats remains unknown, particularly when frozen-thawed semen is used. This study evaluated the distribution of frozen-thawed goat spermatozoa after LAI and compared the effects of sperm numbers and deposition sites (unilateral and bilateral sites) on pregnancy rate. In experiment 1, the frozen-thawed spermatozoa were stained either with CellTracker Green CMFDA (CT-Green) or CellTracker Red CMPTX (CT-Red), and in vitro evaluations of viability and motility were performed. In experiment 2, the labeled spermatozoa were deposited via LAI into the left (CT-Green) and right (CT-Red) uterine horns (n = 4). After ovariohysterectomy (6 hours after insemination), the distributions of green- and red-colored spermatozoa were assessed via tissue section, flushing, and the oviductal contents were also collected. Experiment 3 was designed to test the pregnancy rates in a group of 120 does after LAI using different numbers of spermatozoa (60 and 120 × 10⁶ sperm per LAI) and different deposition sites. The results demonstrated that the fluorochromes used in this study did not impair sperm motility or viability. Frozen-thawed goat spermatozoa can migrate transuterinally after LAI, as evidenced by the observations of both CT-Green– and CT-Red–labeled spermatozoa in both uterine horns. Lower numbers of spermatozoa (60 × 10⁶) that are inseminated unilaterally (either ipsilateral or contralateral to the site of ovulation) can efficiently be used for LAI in goats (with a 56.67% pregnancy rate).