Hormone-induced luteolysis on physiologically persisting corpora lutea in Eurasian and Iberian lynx (Lynx lynx and Lynx pardinus)

The Iberian lynx (Lynx pardinus) is the most critically endangered felid. A high reproductive success within the Iberian Lynx Conservation Breeding Program is crucial to maintaining the goal of reintroducing captive born offspring to the wild and thus increasing the population. Lynx follow a unique reproductive strategy with a monoestrous cycle and persisting CLs over many years. These persistent CLs constantly produce progesterone (on average 5 ng/mL) and are hypothesized to hinder a polyestrous cyclicity in lynx. The aim of this study was to evaluate whether artificial luteolysis can be achieved with common luteolytic drugs and if luteolysis would induce a second estrus naturally. We observed a functional regression of lynx CLs after artificial luteolysis with 2.5 μg/kg body weight PGF2α analogue (cloprostenol) administered three times every 16 hours. We could see a similar effect when combining cloprostenol with other drugs like an anti-gestagen (aglepristone) or a dopamin-agonist (prolactin-inhibitor, cabergolin) or by prolonging the cloprostenol administration to a total of 5 days. However, the sample size was too small to draw conclusions about which protocol is superior or if combining different drugs would result in a positive synergism. Neither structural regression of CLs nor subsequent spontaneous estrus induction was induced with any of these treatments. We suggest that a dose of 2.5 μg/kg body weight cloprostenol administered once daily over 3 to 5 days is sufficient for functional luteolysis in lynx. The next step would be to compare the success of estrus induction with or without the preceding artificial luteolysis.     

Pregnancy diagnosis in urine of Iberian lynx (Lynx pardinus)

Diagnosis of pregnancies is an important management tool for the Iberian lynx Conservation Breeding Program, a program geared to recover the world's most endangered felid. Non-invasive methods such as fecal hormone analyses are not applicable to the lynx, since fecal progestin does not follow the typical pregnancy pattern of felids. Therefore, we aimed to test whether urine can be used as an alternative substance for pregnancy diagnosis in the Iberian lynx.Progesterone immunoreactive metabolites were determined in urine samples of pregnant and non-pregnant females before and during breeding season. Additionally, we used the Witness^®Relaxin test to determine relaxin in blood and urine. No differences were found in progestin concentrations determined in urine samples collected from pregnant and non-pregnant animals between day 1 and 65 following mating. Although the Witness^®Relaxin test was positive in serum samples collected from animals between day 32 and 56 of pregnancy, it failed in both fresh and frozen urine samples collected from the same stage of pregnancy. A weak relaxin reaction in urine samples collected from animals between day 29 and 46 of pregnancy was detectable after urines were concentrated by ultrafiltration (>50×). Concentrated samples obtained from non-pregnant and early pregnant animals yielded negative test results. In conclusion, the Witness^®Relaxin test can be applied for pregnancy diagnosis in Iberian lynx in both serum and concentrated urine samples obtained during the second half of pregnancy. A positive relaxin test indicates an ongoing pregnancy, whereas negative tests must be judged carefully as hormone concentrations might be below detection thresholds.     

Investigations on reproductive physiology in the male Eurasian lynx (Lynx lynx)

This study characterized (in vivo) morphological and functional parameters of reproductive organs of adult male lynx (n = 3) prior to, during, and after the breeding season (n = 3). Size and morphology of the reproductive tract were monitored by transcutaneous (testes) and transrectal (accessory sex glands) ultrasonography. Semen was collected by electroejaculation. Ejaculate volume, sperm number, motility, and morphology of spermatozoa as well as testosterone concentrations in blood serum and feces were evaluated. The testes and prostate had seasonal changes in size and echotexture. The mean (+/- S.D.) maximum and minimum testicular volume were 2.8 +/- 0.8 cm3 and 1.5 +/- 0.3 cm3, respectively. Fecal testosterone concentrations were highest in February (1240 +/- 393 ng/g feces), with a second increase in May (971 +/- 202 ng/g feces), but concentrations were lowest in January (481 +/- 52.9 ng/g feces). Ejaculate volume, total sperm number and percentage of motile, and intact spermatozoa were maximal in March (the middle of the breeding season). In one of the eight litters, multiple paternity was proven; however, in the remaining seven litters, all 16 cubs were sired by the same male. This particular male had the most developed and active testes and best semen quality, which may be important for sperm competition.     

Characterization of reproductive activity in captive male Eurasian lynx (Lynx lynx)

This study characterizes the reproductive activity of male European lynx kept in Central Russia. Four captive adult males were subjected to an electroejaculation prior to (November), during (March) and after (June) the breeding season. Concentration, motility and morphological integrity of spermatozoa as well as testis diameter and testosterone levels in serum were evaluated. Additionally, fecal samples were collected for 2 years to determine the fecal testosterone secretion. Testis sizes and serum testosterone concentrations were characterized by little changes with highest levels in March (2.98 mm³; 1.96 ng/ml testosterone) and lowest in June (2.34 mm³; 0.75 ng/ml testosterone). In faeces, the highest testosterone concentrations were measured in February followed by a second increase in May. The volume of ejaculates and percentages of motile and intact spermatozoa reached the maxima in March. By performing two-male mating experiments, we could prove multiple paternity within three litters. Paternity analysis of litter also revealed that 26 of 31 cubs (84%) were sired from the same male, independently from being the first or second mating partner of the respective female. This particular male showed the most developed and activated reproductive tract and also had the best semen quality, which seems to be important for sperm competition.     

Anatomy and histology of the reproductive tract of the female Babirusa (Babyrousa celebensis)

The anatomy and histology of the female reproductive tract of the Indonesian wild pig Babyrousa celebensis was studied by means of reproductive tracts obtained from seven animals aged between two and 22 years of age. The ovary appeared to have the ability to ovulate up to four ova at one time. However, the combined ovarian output seemed to average 1.86 ova. Ovulation can take place at any time from puberty to old age (22 years). The opening to the uterine tube was indicated by a ‘flower-like' array of tall, broad epithelial ‘petals' arising from the luminal surface of the funnel. The mucosal surfaces of these structures were covered in a mixture of prominent ciliated cells and bulbous secretory cells. The uterine tube followed a tightly convoluted path to the tip of the uterine horn. The uterus was proportionately short. The anatomical construction of the uterus was similar to those of other suids in that the columnar endometrium was heavily folded, there was a rich supply of uterine glands in the lamina propria, and the uterus was provided with a good blood supply. The cervix was thick walled and had a spiral lumen.     

Different cryopreservation requirements in foetal versus adult skin cells from an endangered mammal,the Iberian lynx (Lynx pardinus)

Cryobanking somatic foetal cells acquire much relevance in endangered species for biodiversity conservation purposes. Such cells could be later used to reintroduce the lost genes into the breeding pool, by inducing pluripotency and/or nuclear transfer if necessary. Since requirements for preserving foetal cells are not always the same as for adult ones, we evaluated the cryosensitivity of foetal skin cells in comparison with adult ones from the critically endangered Iberian lynx. Responses to cryoinjury were analyzed in both thawed cell types by means of cell viability and functionality (by analyzing their membrane integrity, metabolic activity, glycosaminoglycan content and proliferative activity). Freezing media included the permeating cryoprotectant Me₂SO, either alone or along with the non-permeating cryoprotectant sucrose at 0.1 or 0.2 M. When Me₂SO was the only cryoprotectant, survival rate fell in thawed foetal cells to 54 ± 4% (against 89 ± 6% for thawed adult ones) and both proliferative and metabolic activities remained significantly lower than values for thawed adult cells. However, the combination of sucrose (both 0.1 as 0.2) and Me₂SO in foetal cells significantly increased their survival rates (to 71 ± 4% and 73 ± 5%, respectively), proliferative activities (partially at day 7 and completely at day 14 after thawing) and metabolic activities.     

Risk taking by Eurasian lynx (Lynx lynx) in a human-dominated landscape: effects of sex and reproductive status

This study aimed to test how the sex and reproductive status of Eurasian lynx influenced their use of 'attractive sinks'– habitats with high prey density and high mortality risks. Locations of 24 Eurasian lynx Lynx lynx were obtained by radio-telemetry in a mixed forest and agricultural habitat in south-eastern Norway. Roe deer, the major food source of lynx in the study area, occurred at higher densities closer to areas of human activity and infrastructure. Proximity of lynx locations to human activity and infrastructure was used as a risk index because the most common causes of death among Scandinavian lynx were of anthropogenic origin. This study shows that distances from lynx locations to human activity were significantly greater for females with newborn kittens than for males, but this decreased with kitten age. The data suggest that this response to human activity is influenced by the reproductive strategies of males and females, and might explain male-biased human-induced mortality in this study and in carnivores more generally.     

Serological survey of Toxoplasma gondii infection in free-ranging Eurasian lynx (Lynx lynx) from Sweden

To investigate the prevalence of Toxoplasma gondii infection in free-ranging Eurasian lynx (Lynx lynx) in Sweden, serosanguinous fluids and feces were collected from 207 carcasses of lynx killed or found dead from 1996 to 1998. Sera were tested for antibodies against T. gondii by the direct agglutination test, and 156 (75.4%) of the sera tested positive at antibody titers>or=40. Antibody prevalence was significantly lower in lynx originating from the northern parts of Sweden than in lynx from the more southern regions that are more densely populated by humans. Age-related differences also were found, with a significantly lower prevalence (55%) in juvenile (<1-yr-old) than in subadult and adult animals (82%). There was no significant difference in seroprevalence between males and females. Oocysts typical of T. gondii were not detected in any of the fecal samples.     

Social play in the development of sibling relations in Eurasian lynx (Lynx lynx)

Social play fulfills an important function in creating and maintaining relations between siblings. However, its relationship with the intralitter social processes is poorly understood. It was noticed that, in Eurasian lynx (Lynx lynx) litters, sex differences in social play are absent in the first 2–3 months of life. It was found that the most intense periods of play behavior (at an age of 9 and 12 weeks) coincide with periods of aggression. Gradual change in play interactions, which require close physical contact by play elements with increased motor activity, are described. This reflects the changes in the relevance of certain skills of lynx cubs as they grow older.     

The reproductive performance of female Formosan sambar deer (Cervus unicolor swinhoei) in semi-domesticated herds

This study documented the reproductive performance of 210 adult female Formosan sambar deer (FSD, Cervus unicolor swinhoei) from four semi-domesticated deer herds in Taiwan. An extensive analysis of 525 reproductive records from 2000 to 2008, including the conditions of estrus, gestation, and parturition was conducted. The mean ± S.E.M. lengths of the estrous cycle, gestation, and fawning interval were 18.2 ± 0.5 d (n = 56), 258.6 ± 0.3 d (n = 160), and 369.9 ± 2.3 d (n = 122), respectively. Hand breeding was performed between June and December (n = 494), with the majority (93.1%) occurring between July and October (P < 0.05). Fawning occurred from February to September (n = 318), and most frequently (83.0%) between April and June (P < 0.05). Pregnancy rate per mating in FSD hinds was 64.4%. There was a 1.3:1 male-to-female ratio at birth (P < 0.05) among 320 fawns, and only two cases of twinning (0.63%). The postnatal mortality rate was 6.6% (21/320), and the mortality rate in fawns before weaning did not exceed 8% on any farm. Fecundity was enhanced by high pregnancy rates and high offspring survival rates. This study provides baseline information on reproductive performance of FSD, which should be valuable to veterinarians and deer industry personnel for management of FSD on farms in subtropical countries.     

Reproductive performance of prepubertal Bos indicus heifers after progesterone-based treatments

The objective was to evaluate the effects of exogenous progesterone (P4) on reproductive performance of prepubertal Bos indicus heifers. Prepubertal Nelore heifers (n = 589; 24.0 ± 1.13 mo; 298.0 ± 1.89 kg; body condition score of 3.2 ± 0.26; mean ± SEM) were randomly assigned to receive, between experimental Days −12 and 0: no treatments (CIDR0; n = 113); a new intravaginal insert (CIDR) containing 1.9 g of P4 (CIDR1; n = 237); or a similar insert previously used three times, with each use occurring for 9 d (CIDR4; n = 239). An additional treatment group was pubertal heifers given 12.5 mg dinoprost tromethamine im on Day 0 (PGF; n = 346), and used as controls for evaluation of conception rates. On Day 0, transrectal palpation was done for uterine score evaluation (UtS; 1-3 scale), blood samples were taken for serum P4 concentrations, and follicle diameter (FD) was measured. The breeding season started on Day 1 and consisted of AI after detection of estrus between Days 1 and 45, and exposure to bulls between Days 46 and 90. There were effects of treatment (P < 0.05) on serum concentrations of P4 on Day 0 (0.37 ± 0.16, 2.31 ± 0.11, and 1.20 ± 0.11 ng/mL for CIDR0, CIDR1, and CIDR4, respectively; mean ± SEM), FD on Day 0 (9.45 ± 0.24, 9.72 ± 0.17, and 11.42 ± 0.16 mm), UtS on Day 0 (1.49 ± 0.06, 1.88 ± 0.04, and 2.24 ± 0.04), estrus detection rates at 7 d (19.5, 42.6, and 38.3%) and 45 d (52.2, 72.1, and 75.3%) of the breeding season, and on pregnancy rates at 7 d (5.3, 14.3, and 18.4%), 45 d (27.4, 39.2, and 47.7%) and 90 d (72.6, 83.5, and 83.7%) of the breeding season. Conception rate 7 d after the start of the breeding season was greater (P < 0.05) in heifers from the CIDR4 (46.8%) and PGF (43.8%) groups than in the CIDR0 (27.3%) and CIDR1 (33.7%) groups. In conclusion, exogenous P4 hastened puberty and improved pregnancy rates at the beginning of the breeding season in prepubertal Bos indicus heifers. Furthermore, previously used CIDR inserts were better than new inserts.     

Two-trait random regression model to estimate the genetic association of scrotal circumference with female reproductive performance in Nelore cattle

In an attempt to determine when scrotal circumference (SC) could be a reasonable indicator of female reproductive performance, a series of two-trait random regression model (regression for SC on age at measurement) using Gibbs sampling was applied to field data of Nelore cattle raised in a tropical environment. The female traits evaluated were age at first calving (AFC), first calving interval (FCI), heifer pregnancy (HP), and stayability (STAY). The posterior means of heritability of female traits ranged from 0.15 for AFC to 0.46 for HP and were about 0.50 for SC. The posterior means of genetic correlations between SC and AFC, FCI, HP, STAY were up to −0.70, −0.25, 0.48, and 0.29, respectively. Genetically, SC could be a reasonable indicator of female puberty (e.g., HP) as long as it is measured at a young age (400–440 days). However, for female reproductive traits measured at an older age (e.g., STAY), SC is merely a modest or even poor indicator. The use of sire expected progeny differences for female reproductive traits will be more effective than the use of expected progeny differences for SC to improve the reproductive performance of female cattle.     

Measuring fecal progestogens as a tool to monitor reproductive activity in captive female bottlenose dolphins (Tursiops truncatus)

The objective was to develop and test radioimmunoassays (RIAs) to measure fecal progestogens (P) and estrogens (E) to monitor ovarian activity in the bottlenose dolphin (Tursiops truncatus). Fecal samples were collected at least once a week for 20 mo from three peripubertal female bottlenose dolphins. Blood samples were collected at least once a month to compare serum and fecal steroid concentrations. Moreover, random fecal samples from three pregnant females, one lactating female, and one sexually mature female receiving oral altrenogest treatment were also collected. Fecal samples were collected behaviorally with a probe to avoid water contamination and extracted with petroleum ether (for P analysis) or diethyl ether (for E analysis). When possible, vaginal cytology and ovarian ultrasonography were used to monitor the estrous cycle. The RIA for fecal P had good reproducibility and negligible matrix effect. In addition, when fecal samples (N = 25) were extracted with ethanol, the results with the two methods of extraction were highly correlated (r = 0.923). Therefore, extraction of fecal samples with petroleum ether represented a valid alternative to other, more time-consuming methods of determining fecal P concentrations. In the absence of luteal activity, fecal P concentrations were consistently < 10 pmol/g feces, although they never decreased below 10 pmol/g during pregnancy. Thus, the threshold to confirm the presence of an active corpus luteum was provisionally set at 10 pmol/g. Around the onset of puberty, luteal phases appeared shorter and irregular in the bottlenose dolphin, as in other mammalian species. Additional HPLC-MS studies should be performed to identify predominant P metabolites to be used as fecal indicators of luteal activity in this species.     

Spatial density estimates of Eurasian lynx (Lynx lynx) in the French Jura and Vosges Mountains

Obtaining estimates of animal population density is a key step in providing sound conservation and management strategies for wildlife. For many large carnivores however, estimating density is difficult because these species are elusive and wide‐ranging. Here, we focus on providing the first density estimates of the Eurasian lynx (Lynx lynx) in the French Jura and Vosges mountains. We sampled a total of 413 camera trapping sites (with two cameras per site) between January 2011 and April 2016 in seven study areas across seven counties of the French Jura and Vosges mountains. We obtained 592 lynx detections over 19,035 trap days in the Jura mountains and 0 detection over 6,804 trap days in the Vosges mountains. Based on coat patterns, we identified a total number of 92 unique individuals from photographs, including 16 females, 13 males, and 63 individuals of unknown sex. Using spatial capture–recapture (SCR) models, we estimated abundance in the study areas between 5 (SE = 0.1) and 29 (0.2) lynx and density between 0.24 (SE = 0.02) and 0.91 (SE = 0.03) lynx per 100 km². We also provide a comparison with nonspatial density estimates and discuss the observed discrepancies. Our study is yet another example of the advantage of combining SCR methods and noninvasive sampling techniques to estimate density for elusive and wide‐ranging species, like large carnivores. While the estimated densities in the French Jura mountains are comparable to other lynx populations in Europe, the fact that we detected no lynx in the Vosges mountains is alarming. Connectivity should be encouraged between the French Jura mountains, the Vosges mountains, and the Palatinate Forest in Germany where a reintroduction program is currently ongoing. Our density estimates will help in setting a baseline conservation status for the lynx population in France.     

Male reproductive traits, semen cryopreservation, and heterologous in vitro fertilization in the bobcat (Lynx rufus)

There is limited information on bobcat ejaculate traits and sperm cryopreservation and fertilizing ability. Bobcats were electroejaculated under general anesthesia in November (autumn) and April (spring), and endocrine and sperm traits were characterized. Testosterone (mean ± SEM: 0.90 ± 0.15 ng/mL) was not different between sampling times, but cortisol (average: 13.95 ± 1.73 μg/dL) was significantly higher in April. Average number of spermatozoa was 10.0 ± 3.4 × 10⁶ sperm/ejaculate, with values being significantly higher in April. Sperm motility (average 55.7 ± 5.8% motile sperm) was not different between sampling times. The proportion of normal spermatozoa in the ejaculate (average: 14.7 ± 2.1%) was significantly higher in April, but the percentage of spermatozoa with intact acrosomes (average: 43.7 ± 3.8%) was significantly higher in autumn. Spermatozoa were cryopreserved in a Tes-Tris-based diluent (TEST) or Biladyl, both containing 20% egg yolk and 4% glycerol. Diluted sperm were loaded into straws, refrigerated using a programmable thermoblock with a dry chamber, frozen in nitrogen vapors, thawed, and incubated in F-10 medium with 5% fetal bovine serum for up to 3 h. After cryopreservation in TEST, there were about 50% motile sperm upon thawing, and survival was high during incubation post-thaw. Cryopreservation in Biladyl led to similar results, but motility decreased substantially during incubation post-thaw. Bobcat spermatozoa fertilized domestic cat oocytes matured in vitro. Fertilization rates were higher for sperm collected in April and cryopreserved in TEST (46%) than for those cryopreserved using Biladyl (<3%). Fertilized oocytes cleaved in culture, and some (27%) reached the morula stage. This study has allowed us to gain further baseline information on bobcat reproduction, explore sperm cryopreservation conditions, and show that fertilizing capacity can be tested using in vitro-matured cat oocytes. These results will be important for future conservation efforts.