Survey among FELASA members about rehoming of animals used for scientific and educational purposes.

Rehoming is an important fate, which should be considered for animals used for scientific and educational purposes, and which is highlighted in the European Directive 2010/63 EU. In 2018, the Federation of European Laboratory Animal Science Associations (FELASA) convened a working group to review current literature and identify existing practices with the aim of issuing general recommendations on the rehoming of research animals. In order to understand the number and species of animals being rehomed and which species and information to include in the recommendations, the working group launched a survey that was distributed among FELASA members, yielding 97 valid records for analysis. Most respondents of the survey considered the rehoming of cats, dogs, mice, rats, rabbits, pigs and minipigs. The most important issues reported by the respondents were related to availability/suitability of animals, availability of adopters and legal issues. Based on the data and information collected in this survey, the working group decided on the format and content of the future recommendations: a first section containing a general protocol for rehoming, addressing the issues raised by the respondents, and a second section containing species-specific information and advice about cats, dogs, small prey mammals, equines, primates, camelids and minipigs.

FELASA recommendations for the rehoming of animals used for scientific and educational purposes.

Directive 2010/63/EU of the European Parliament and the Council of 22 September 2010 states that at the end of a procedure, the most appropriate decision on the future of an animal previously used or intended for use in scientific procedures should be taken on the basis of animal welfare and potential risks to the environment. Member States may allow animals to be rehomed provided the health of the animal allows it, there is no danger to public health, animal health or the environment and if appropriate measures have been taken to safeguard the wellbeing of the animal. In countries where rehoming is permitted, it is the responsibility of the Animal Welfare Body to advise on a rehoming scheme which must include appropriate socialization in order to help facilitate successful rehoming, avoid unnecessary distress to the animals and guarantee public safety. This paper reviews the EU legislation, existing guidance, current literature and best practice to define rehoming, sets out general considerations for rehoming laboratory animals including socialization and provides practical advice on the steps required in a rehoming scheme. For those species most frequently rehomed, more detailed species-specific sections are included.

Long-acting reversible contraception with etonogestrel implants in female macaques (Macaca mulatta and Macaca fascicularis).

Introduction: Contraception is often required for management and population control purposes in group-housed and free-roaming non-human primates. Long-acting reversible contraceptives, including subdermal progestin-releasing implants, are preferred as they eliminate challenges associated with frequent administration. Etonogestrel (ENG)-releasing subdermal implants are reversible and long-acting for a minimum of 3 years, and are commercially available for human use as Implanon® or Nexplanon®. Methods: A retrospective analysis was performed detailing the contraceptive effectiveness and reversibility of subdermal placement of one-fourth or one-third of an ENG implant (68 mg/implant) in 129 female rhesus macaques (Macaca mulatta) and 67 cynomolgus macaques (Macaca fascicularis) at the Biomedical Primate Research Centre (Rijswijk, Netherlands). Furthermore, single cross-sectional ENG serum concentrations were measured for 16 rhesus and 10 cynomolgus macaques, and hemoglobin and blood chemistry pre-ENG and at timepoints >0.5, >1.5, and > 2.5 years post-ENG insertion were evaluated for 24 rhesus macaques. Finally, data were obtained using trans-abdominal ultrasound regarding the influence of ENG on uterine volume and endometrial thickness in 14 rhesus and 11 cynomolgus macaques. Results: As a contraceptive ENG was in 99.80% (CI 93.50-99.99) and 99.95% (CI 99.95-100) effective in rhesus and cynomolgus macaques, respectively. Prolonged ENG durations of implant use in 14 rhesus macaques (range 3.1-5.0 years) and eight cynomolgus macaques (range 3.2-4.0 years) resulted in no unintended pregnancies. A total of 17 female macaques were allowed to breed after ENG removal, and among them, 14 female macaques (82%) had an uneventful delivery. Serum ENG concentrations with a median ENG duration of 1.2 years (range 0.1-6.0 years) and 1.9 years (range 0.6-4.7 years) resulted in median concentrations of 112 pg./mL (range 0-305 pg./mL) and 310 pg./mL (range 183-382 pg./mL) for rhesus and cynomolgus macaques, respectively. ENG had no clinical effect on hemoglobin and blood chemistry parameters nor on the thickness of the endometrial lining or uterus volume. Conclusion: This study indicates that both one-fourth and one-third of the ENG implants are effective, long-acting, reversible, and safe contraceptive to use in macaques.

Multigenerational Social Housing and Group-Rearing Enhance Female Reproductive Success in Captive Rhesus Macaques (Macaca mulatta).

To optimize costs and reproductive success, rhesus macaques in biomedical primate research facilities are often peer-reared. Older, dependent infants are typically removed from their natal group to enhance female reproduction. The minimal husbandry age-norm of infant removal is ten months. These practices deviate from species-specific behavior and may reduce welfare, suggesting a trade-off between female reproduction and welfare. However, the effect of breeding group type and rearing history on female reproductive success (i.e., birth rate; inter-birth interval (IBI); offspring survival) is unclear. This retrospective study investigated whether group type (i.e., peer groups versus multigenerational groups) and rearing history (i.e., peer- or hand-reared; group-reared with peer- or hand-reared mother; group-reared) affected female reproductive success in captive rhesus macaques. Data on female reproduction between 1996 and 2019 were collected at the Biomedical Primate Research Centre, Rijswijk. Birth rates were higher in multigenerational breeding groups than in peer groups. Moreover, group-reared females had higher offspring survival than peer- or hand-reared females. IBI was not affected by breeding group type or female rearing history. However, females in both peer and multigenerational breeding groups typically conceived earlier after giving birth than the husbandry infant removal age-norm of ten months. Thus, infant removal at an age of ten months does not enhance a female's reproduction. Altogether, female reproduction and non-human primate welfare can simultaneously be optimized through multigenerational breeding groups and group-rearing.

The Older the Better: Infanticide Is Age-Related for Both Victims and Perpetrators in Captive Long-Tailed Macaques.

In wild primates, infanticide is a risk that is especially prevalent when a new male takes over the alpha position. Insight into risk factors related to infanticide may decrease the incidence of infanticide in captivity during male introductions. We investigated several risk factors of infanticide derived from hypotheses explaining infanticide in the wild and tested this in captive long-tailed macaques (Macaca fascicularis) using demographic data spanning a 25.5-year period. Factors that are related to infanticide in the wild explained a large proportion, but not all incidences, of infanticide in captivity. Consistent with the wild data, infants young enough to decrease the interbirth interval (<215 days) were at risk of being killed. In contrast to studies from the wild, infanticidal males were more than 2.5 years younger than non-infanticidal males. This indicates that captive settings can lead to new risks since relatively young males may gain the alpha position, promoting infanticide. Therefore, we propose the adolescent male risk hypothesis as a captive risk factor in which subadult males pose a risk of infanticide. In conclusion, the ages of both males and infants are related to infanticide in captivity and have to be taken into account during male introductions.

Overweight Management through Mild Caloric Restriction in Multigenerational Long-Tailed Macaque Breeding Groups.

Caloric restriction (CR) is an effective method to reduce overweight in captive non-human primates (NHPs). CR has been applied to individually- and pair-housed NHPs, but whether applying CR can be effective and safe in group-housed NHPs has not yet been assessed. This study investigates the effect of mild (20%) CR on adult overweight and biochemical parameters, immature growth, veterinary consultations, and reproductive success in multigenerational long-tailed macaque (Macaca fascicularis) breeding groups. Data were derived from anthropometric measurements and blood samples during yearly health checks, complemented with retrospective data on veterinary consultations and reproductive success. Adult body measures decreased after CR, with heavier individuals and females losing more weight compared to leaner individuals and males. CR lowered cholesterol levels in adults but had no overall effect on other biochemical parameters. Yet, biochemical parameters of individuals with high baseline values were reduced more compared to individuals with low baseline values. Immature growth, veterinary consultations and reproductive success were not influenced by CR. Thus, CR targeted the right individuals, i.e., overweight adults, and had no adverse effects on the variables examined in this study. This implies that mild CR can be a valuable overweight management strategy in group-housed NHPs.

Dynamic evolution of Mhc haplotypes in cynomolgus macaques of different geographic origins.

The major histocompatibility complex (MHC) plays a key role in immune defense, and the Mhc genes of cynomolgus macaque display a high degree of polymorphism. Based on their geographic distribution, different populations of cynomolgus macaques are recognized. Here we present the characterization of the Mhc class I and II repertoire of a large pedigreed group of cynomolgus macaques originating from the mainland north of the isthmus of Kra (N = 42). Segregation analyses resulted in the definition of 81 unreported Mafa-A/B/DRB/DQ/DP haplotypes, which include 32 previously unknown DRB regions. In addition, we report 13 newly defined Mafa-A/B/DRB/DQ/DP haplotypes in a group of cynomolgus macaques originating from the mainland south of the isthmus of Kra/Maritime Southeast Asia (N = 16). A relatively high level of sharing of Mafa-A (51%) and Mafa-B (40%) lineage groups is observed between the populations native to the north and the south of isthmus of Kra. At the allelic level, however, the Mafa-A/B haplotypes seem to be characteristic of a population. An overall comparison of all currently known data revealed that each geographic population has its own specific combinations of Mhc class I and II haplotypes. This illustrates the dynamic evolution of the cynomolgus macaque Mhc region, which was most likely generated by recombination and maintained by selection due to the differential pathogenic pressures encountered in different geographic areas.

Effect of Housing Conditions on Cortisol and Body Fat Levels in Female Rhesus Macaques.

Macaques are among the most commonly used non-human primates in biomedical research. They are highly social animals, yet biomedical studies often require group-living animals to be pair-housed in a controlled environment. A change in environment causes only short-term stress in adapting individuals, while non-adapting animals may experience long-term stress that can adversely affect study results. Individuals likely differ in their ability to adapt depending on individual characteristics. Changes in cortisol and body fat levels may reflect these different individual responses. Here, we investigate the long-term effect of a change from group- to pair-housing on cortisol and body fat levels in 32 female rhesus macaques, exploring whether age, dominance rank, original cortisol, and body fat levels are related to long-term stress in pair-housing. Hair samples were analyzed for cortisol levels, while anthropometric measurements and computed tomography were performed to quantify body fat. Monkeys served as their own control with a 7.5-month period between the measurements. Cortisol levels increased, while average body fat levels did not differ when individuals were moved from group- to pair-housing. Cortisol and body fat levels were not significantly correlated. Changes in cortisol were independent of age and dominance rank, whereas individual variation in body fat alterations was related to the group-housed body fat level and dominance rank. Although this study did not identify individual characteristics related to long-term stress in pair-housing, the individual variation confirms that some individuals are more resilient to change than others and provides possibilities for future refinement studies.

Retrospective Analysis of the Effectiveness and Reversibility of Long-Acting Contraception Etonogestrel (Implanon®) inCommon Marmosets (Callithrix jacchus).

Contraception is an important population control method for the colony management of primates housed in captivity. Etonogestrel (ENG) implants (i.e., Implanon®) are a widely used progestin-based contraceptive in common marmosets (Callithrix jacchus) with the theoretical advantages of being reversible and long-acting. However, no dose and efficacy data are available yet. Therefore, data from 52 adult female marmosets contracepted with ENG (one-fourth or one-third of an implant) housed at the Biomedical Primate Research Centre (BPRC, Rijswijk, The Netherlands) over the past 18 years were analyzed. Using an electronic database, a retrospective longitudinal cohort study was conducted to calculate the reproductive data before, during and after ENG use. The data show an effectiveness in preventing pregnancy of 99%. The implant was effective within one week after insertion. Unintended pregnancies did occur, but in 60% of these cases, the animals were already pregnant at the time of implant insertion. In these cases, healthy offspring were born despite the use of the implant. No stillbirths, neonatal deaths or maternal deaths could be linked to ENG use. After implant removal, 83% of the animals delivered healthy offspring. No difference in contraception efficacy was observed between the use of one-fourth or one-third of an implant. ENG achieved a contraceptive protection exceeding 99% and was shown to be reversible concerning fertility. To our knowledge, this is the first detailed analysis on the use of ENG in marmosets.

A Stepwise Male Introduction Procedure to Prevent Inbreeding in Naturalistic Macaque Breeding Groups.

Male introductions into captive primate breeding groups can be risky and unsuccessful. However, they are necessary to prevent inbreeding in naturalistic breeding groups. The procedure used to introduce new individuals may affect the success and influence the risks associated with group introductions. At the Biomedical Primate Research Centre (BPRC) in Rijswijk, the Netherlands, male rhesus macaque (Macaca mulatta) introductions into naturalistic social groups with a matrilineal structure and without a breeding male achieve relatively high success rates. This paper describes the male introduction procedure used at the BPRC. Males are stepwise familiarized with and introduced to their new group, while all interactions between the new male and the resident females are closely monitored. Monitoring the behaviour of the resident females and their new male during all stages of the introduction provides crucial information as to whether or not it is safe to proceed. The BPRC introduction procedure is widely applicable and may improve the management of captive primate groups in any housing facility worldwide. Thus, the careful introduction management can minimize the risk associated with male introductions and enhance the welfare of captive primates.

Factors determining male introduction success and long-term stability in captive rhesus macaques.

The entrance of new males into non-human primate groups bears high social risk, yet migration is necessary to prevent inbreeding. Males are not always accepted in their new group. In the wild, males may increase the likelihood of successful group entry by choosing a new group based on their own and the group's characteristics. Understanding whether these characteristics also determine a male's ability to enter captive groups is crucial to improve introduction management. This study aims to identify which factors determine male introduction success (i.e. male stays in the group for at least 4 weeks) and long-term stability (i.e. the male does not cause considerable behavioural problems after success) after male introductions in captive groups of rhesus macaques (Macaca mulatta), creating one-male groups. We studied 64 male introductions at the breeding colony of the Biomedical Primate Research Centre in Rijswijk, The Netherlands. 49 (77%) introductions were successful, with the male obtaining a long-term stable social position in the group in 38 (59%) introductions. Introductions of males that reached at least prime age, into groups with more adult females, but without pregnant females were most successful. Moreover, long-term stability was highest when males were heavier, were at least 3.5 years old when they were first removed from their natal group, and groups had few matrilines and no pregnant females were present. Males should be introduced at the time they would naturally immigrate, when they are strongest. Moreover, groups should consist of few large matrilines, as observed in the wild, with philoatric females and males that are removed at natural age. Our study highlights the importance of composing naturalistic groups and mimicking natural migration patterns to maintain long-term stable breeding groups in captivity.

Defining predictive factors for reproductive output in captive common marmosets (Callithrix jacchus).

Common marmosets (Callithrix jacchus) demonstrate variations in reproductive output, not only in terms of total reproductive output during a lifetime but also in litter size per parturition. The present study explores factors, such as parents' litter size, parturition number, maternal body weight at conception and maternal age, which may account for this variation. A retrospective analysis of clinical records of a captive breeding colony was conducted over a 9-year period yielding reproductive summaries of 26 dams and 22 sires producing a total of 115 litters. Dams born from litters of ≤2 (N = 20) more often produced litters of ≤2, whereas dams born from litters of >2 (N = 6) more often produced litters of >2 (p < 0.05). The dams' maternal body weight at the time of conception had also a significant effect on subsequent litter size. In addition, the chance of triplets was higher after the second parturition. Maternal age, interbirth interval, and season of birth had no effect on litter size. Factors relating to the sire had a negligible effect on the size of the litter. Multivariate statistical modeling revealed that the dams' original litter size, maternal bodyweight at conception and parturition number are determining factors for the number of babies per litter. This study identified factors determining marmoset litter size, some of which (maternal litter size) are novel to this study and were not reported previously. Further exploration of the potential role of maternal litter size as a determinant of the litter sizes produced by marmoset breeders is warranted.

Evaluation of ultrasonic vocalizations in common marmosets (Callithrix jacchus) as a potential indicator of welfare.

The vocal repertoire in common marmosets (Callithrix jacchus) has been assumed to consist not only of vocalizations audible to humans but also of ultrasonic vocalizations (USVs). The use of USVs to socially indicate distress has not been evaluated in this species, however. The authors analyzed the ultrasonic vocal repertoire of the common marmoset under normal housing conditions, under various experimental manipulations intended to elicit positive or negative emotional responses and during stressful experiences including blood draw and exposure to a perceived predator. Analysis of the recordings showed that marmosets produced vocalizations with ultrasonic components as part of their normal vocal repertoire, but these vocalizations all have audible components as well. Only 4 of the 13 types of vocalizations had ultrasonic components. These ultrasonic components were not reliably associated with responses to different experimental manipulations, suggesting that they are not used to indicate pain, discomfort or distress.

The behavioral context of visual displays in common marmosets (Callithrix jacchus).

Communication is important in social species, and may occur with the use of visual, olfactory or auditory signals. However, visual communication may be hampered in species that are arboreal have elaborate facial coloring and live in small groups. The common marmoset fits these criteria and may have limited visual communication. Nonetheless, some (contradictive) propositions concerning visual displays in the common marmoset have been made, yet quantitative data are lacking. The aim of this study was to assign a behavioral context to different visual displays using pre-post-event-analyses. Focal observations were conducted on 16 captive adult and sub-adult marmosets in three different family groups. Based on behavioral elements with an unambiguous meaning, four different behavioral contexts were distinguished: aggression, fear, affiliation, and play behavior. Visual displays concerned behavior that included facial expressions, body postures, and pilo-erection of the fur. Visual displays related to aggression, fear, and play/affiliation were consistent with the literature. We propose that the visual display "pilo-erection tip of tail" is related to fear. Individuals receiving these fear signals showed a higher rate of affiliative behavior. This study indicates that several visual displays may provide cues or signals of particular social contexts. Since the three displays of fear elicited an affiliative response, they may communicate a request of anxiety reduction or signal an external referent. Concluding, common marmosets, despite being arboreal and living in small groups, use several visual displays to communicate with conspecifics and their facial coloration may not hamper, but actually promote the visibility of visual displays.

Multilocus definition of MHC haplotypes in pedigreed cynomolgus macaques (Macaca fascicularis).

Cynomolgus macaques (Macaca fascicularis) are used widely in biomedical research, and the genetics of their MHC (Mhc-Mafa) has become the focus of considerable attention in recent years. The cohort of Indonesian pedigreed macaques that we present here was typed for Mafa-A, -B, and -DR, by sequencing, as described in earlier studies. Additionally, the DRB region of these animals was characterised by microsatellite analyses. In this study, full-length sequencing of Mafa-DPA/B and -DQA/B in these animals was performed. A total of 75 different alleles were observed; 22 of which have not previously been reported, plus 18 extended exon 2 alleles that were already known. Furthermore, two microsatellites, D6S2854 and D6S2859, were used to characterise the complex Mafa-A region. Sequencing and segregation analyses revealed that the length patterns of these microsatellites are unique for each Mafa-A haplotype. In this work, we present a pedigreed colony of approximately 120 cynomolgus macaques; all of which are typed for the most significant polymorphic MHC class I and class II markers. Offspring of these pedigreed animals are easily characterised for their MHC by microsatellite analyses on the Mafa-A and -DRB regions, which makes the cumbersome sequencing analyses redundant.

Extensive sharing of MHC class II alleles between rhesus and cynomolgus macaques.

In contrast to rhesus monkeys, substantial knowledge on cynomolgus monkey major histocompatibility complex (MHC) class II haplotypes is lacking. Therefore, 17 animals, including one pedigreed family, were thoroughly characterized for polymorphic Mhc class II region genes as well as their mitochondrial DNA (mtDNA) sequences. Different cynomolgus macaque populations appear to exhibit unique mtDNA profiles reflecting their geographic origin. Within the present panel, 10 Mafa-DPB1, 14 Mafa-DQA1, 12 Mafa-DQB1, and 35 Mafa-DRB exon 2 sequences were identified. All of these alleles cluster into lineages that were previously described for rhesus macaques. Moreover, about half of the Mafa-DPB1, Mafa-DQA1, and Mafa-DQB1 alleles and one third of the Mafa-DRB exon 2 sequences are identical to rhesus macaque orthologues. Such a high level of Mhc class II allele sharing has not been reported for primate species. Pedigree analysis allowed the characterization of nine distinct Mafa class II haplotypes, and seven additional ones could be deduced. Two of these haplotypes harbor a duplication of the Mafa-DQB1 locus. Despite extensive allele sharing, rhesus and cynomolgus monkeys do not appear to possess identical Mhc class II haplotypes, thus illustrating that new haplotypes were generated after speciation by recombination-like processes.