The aims of this work were (1) to characterize the proteome profile of Saimiri collinsi (Amazon squirrel monkey) sperm, and (2) to evaluate if there are meaningful differences in sperm quality and sperm protein landscape during the non-fatted and fatted conditions of the animals. Semen of adult males (n=4) was collected by electroejaculation and diluted in ACP-118® extender. Sperm concentration, motility, vigor, plasma membrane integrity and functionality, and sperm morphology were evaluated. Sperm proteome was characterized by mass spectrometry. The non-fatted and fatted conditions were defined according to the fattening index. There were increases in seminal volume, pH, and sperm motility of S. collinisi during the fatted in comparison with the non-fatted condition (p < 0.05). Animals in the fatted condition had greater body mass (909 ± 23.98 g) in comparison with the non-fatted condition (818.8 ± 5.97 g, p = 0.0001). There were 1161 proteins identified in the sperm of the monkeys. Thirty-seven proteins were upregulated (p < 0.05) in non-fatted condition (such as coiled-coil domain-containing protein 105, protein kinase C alpha type isoform X1, pyruvate dehydrogenase protein X component – mitochondrial and heat shock 70 kDa protein 1-like) and they were involved in cellular and metabolic processes, catalytic activity and binding. Twenty-eight sperm proteins proteins were upregulated (p < 0.05) in fatted condition (such as Cytochrome b-c1 complex subunit 2, mitochondrial; calcium-binding mitochondrial carrier protein SCaMC-1-like; heat shock 70 kDa protein 13, and izumo sperm-egg fusion protein 1), participating in cellular processes, metabolic processes, and localization, binding, catalytic activity, ATP-dependent activity, and transport activity. Functional annotations of sperm proteins upregulated in non-fatted condition were described as hydrogen ion transport, chaperone, cilium, flagellum, and cell projection. In in fatted conditions, such annotations were described as fertilization, spermatogenesis and transport, chaperone, and motor protein. The present study brings an unprecedent contribution to our understanding about the reproductive biology of adult Saimiri collinsi. It also provides support for conservation and development of assisted reproductive technologies for Neotropical primates.
Reproductive activity in animals is regulated by variations in plasma levels of steroid hormones, which respond to both geophysical and social-environmental cues. Changes in testosterone (T) levels play a crucial role in coordinating the morphological, physiological, and behavioral adaptations among males, orchestrating gonadal recrudescence and regression in response to seasonal shifts in environmental factors. Although these reproductive neuroendocrine processes have been extensively examined in various bird species, they remain largely unexplored in owls. The Tropical Screech Owl is widely distributed across Central and South America, and it exhibits regional variations in the timing of chick hatching. This study hypothesized that male Tropical Screech Owls (Megascops choliba) exhibit fluctuations in testosterone levels and corresponding seasonal expression of reproductive genes, potentially linked to reproductive activity, including the presence of semen. To investigate this seasonal reproduction, a group of seven adult male owls was monitored for 14 consecutive months under ex-situ conditions. These owls underwent cloacal electrostimulation and subsequent massage to collect semen, as well as blood sampling to measure serum T levels. The expression of aromatase (CYP19A1), androgen receptor (AR), and estrogen receptor (ESR1 and ESR2) genes was also assessed in gonadal and hypothalamic tissues from four reproductively active and four non-reproductive males collected in-situ from a wild population. Serum T levels in males were significantly higher in July and August, coinciding with the presence of sperm. No significant difference was observed in the hypothalamic tissue transcripts of reproductive versus non-reproductive males. However, non-reproductive males exhibited higher expression of AR and ESR1 genes at the gonadal level. These results confirm the reproductive seasonality of M. choliba males during the winter season in São Paulo State.
The ex situ polar bear population in the United States is not sustainable. Current management practices include pairing mates at five to six years of age, but there is evidence that wild bears have sired cubs as early as two years old. Timing acquisition of sexual maturity in male polar bears could aid in determining when mates should be paired in ex situ populations. To address this, non-invasive fecal testosterone metabolite monitoring was evaluated as a technique for establishing age of sexual maturity in 14 male polar bears (aged 1–6.9 years) for 1–6 years. Testosterone metabolite concentrations were monitored throughout the year and evaluated by breeding season (Jan. 1 – May 21) compared to non-breeding season (May 22 – Dec. 31). Sexual maturity was defined as when testosterone metabolite concentrations were significantly greater during breeding season than non-breeding season and bi-weekly averages were elevated above baseline for a minimum of ten weeks during breeding season mimicking patterns in adult male polar bears. Testosterone metabolite concentrations ranged from 5.9 to 8342 ng/g. One bear met the defined criteria at two years of age and an additional three bears met criteria at three years old. Due to the sampling strategy and the requirements set forth by the definition of sexual maturity, there were confounding results. In two cases, bears determined to be sexually mature the year prior, were not categorized as mature the next year. Another bear demonstrated greater testosterone metabolite values during the non-breeding season than breeding season. This study demonstrated the variability in fecal testosterone metabolites in juvenile polar bears and results suggest that hormone assessment alone is not adequate for determining sexual maturity in ex situ male polar bears. However, if used in tandem with behavioral monitoring and semen collection, when possible, could aid breeding management decisions for ex situ populations.
Seasonal reproduction can provide species with fitness advantages by allowing the birth of young to coincide with favorable environmental conditions, particularly in environments with high seasonal and intra-annual variation in these conditions. Seven of the eight species in the bear family (Ursidae) reproduce seasonally in both managed care and the wild; however, data for the eighth species, the sun bear (Helarctos malayanus), is unclear. Sun bears have been observed to reproduce throughout the year in managed care, yet currently there are no clear data of birth timing for wild sun bear cubs. Here we investigate the seasonality of parturition of wild sun bears by utilizing a dataset of body mass measurements from bears placed in the care of the wildlife conservation organization Free the Bears in Cambodia after interception by authorities for illegal removal from the wild. We selected body mass records for all rescued bears ≤5 kg, and modeled growth rates with linear regressions for cubs with >3 measurements. Assuming a mass of 300 g at birth, these growth rates were used to estimate the birth date for each cub. Our results suggest that wild sun bears reproduce aseasonally in Cambodia, with estimated birth dates occurring in all 12 months of the year, and in all 4 of Cambodia’s distinct seasons.
Dasyprocta fuliginosa had a gestational period of approximately 104 days, to date there are few descriptions of fetal development in this species. From animals collected through subsistence hunting in the Amazon, it was possible to follow the fetal development of the female urogenital apparatus of animals from 56 days until the end of gestation. The kidneys of younger fetuses showed discreet lobations, and from the 75th gestational day onwards they were defined as smooth unilobed organs. The glomerular composition of the cortical region of the youngest fetuses had morphological differences, with poorly developed and undifferentiated capillaries. This composition was transmuted around the 82nd gestational day. Throughout the fetal development process the ureters inserted dorsolaterally to the urinary bladder, communicating with the fetal umbilical funiculus through the urachus, and with the urethra. The same occurred with the reproductive system, which was composed of a double-type uterus, with two uterine horns, a septum and two individual cervical canals, in addition to two uterine tubes, two ovaries, a vagina with a semi-open vaginal membrane, and a vulva, which made up the external genitalia. This constitution reveals that D. fuliginosa during the fetal phase has a well-developed morphology of the reproductive and urinary systems.
Gamete preservation of wild species is a priority in conservation programs. However, field techniques to recover sperm from deceased animals may impair its final quality.
The cryopreservation process also has its challenges, and the minimal required quality parameters may differ depending on the intended use of the semen.
Often, postmortem harvested and cryopreserved sperm may need to undergo a selection process to improve the final quality of the sample. Certain sperm selection techniques that employ centrifugal forces can increase the risk of damaging the cells. In contrast, microfluidics uses hydrostatic pressure and capillary forces, avoiding the need for costly equipment and minimizing cell damage.
This study aims to compare sperm motion parameters, morphology, and acrosome integrity of cryopreserved-thawed epididymal bighorn ram sperm using two commercial selection techniques, VetCount Harvester® device (MFD) and Bovipure™ (NidaCon International AB, Möndal, Sweden) density gradient centrifugation (DGC).
Sperm parameters associated with quality were, progressive motile sperms (PMS), straight velocity (VSL), average path velocity (VAP), curvilinear velocity (VCL), linearity (LIN), straightness (STR), beat cross frequency (BCF), amplitude of lateral head deviation (ALH) and plasma membrane integrity (PMI). Computer Assisted Sperm Analyzer was used for the semen analysis. Eosin/nigrosine and Spermac stains were used to assess morphology and acrosome integrity, respectively. Two veterinarians counted the total number of morphologically normal sperm with complete acrosome.
Mean sperm concentration was higher (P < 0.01) before filtration (129.56 ±53.9 ×106/mL) than DCG (68.42 ±29.18 x106/mL) and MFD (52.94 ±28.76 ×106/mL). PMS was higher (P <0.01) after selection using the DGC (79.70 ± 11.11) and MFD (81.23 ±5.64) compared to the no sperm-selected controls (45.36 ±14.67). Sperm kinetics parameters (VCL, VSL, VAP, BCF, LIN, and STR) were higher in the DGC (P<0.01). However, ALH (P<0.01) was significantly higher in the MFD and Control. Morphological normal sperm, acrosome integrity, and plasma membrane integrity were higher (P<0.01) in samples processed with DGC and MFD.
In conclusion, microfluidics and single-layer centrifugation improved motion parameters, viability, and proportion of morphologically normal sperm with intact acrosomes from frozen-thawed sperm harvested from Bighorn epididymis. The VetCount Harvester® microfluidic device was comparable to DGC and could be an alternative option for sperm selection. Further research is needed to explore the effect of selected sperm using this technique on artificial insemination, re-cryopreservation, and fertility outcomes.
Japan arcs from north to south and is abundant in biodiversity with different climates in each region. Japan has many endemic species/populations, such as the Tsushima leopard cat (Prionailurus bengalensis euptilurus) and the Okinawa rail (Hypotaenidia okinawae), which threaten to become endangered. Furthermore, Japan is one of the countries with the highest number of zoos and aquariums in the world and keeps many wildlife species, many of which are endangered, in captive environments. Effective management of genetic diversity and acceleration of reproduction are essential for the conservation of endangered species, both inside and outside their natural habitats. Germ cell preservation and assisted reproductive technologies have great potential to rescue genetic resources and contribute to the reproduction of endangered species. The long-term preservation of germ cells in Japan is reviewed here in the context of the biodiversity maintenance of germ cells from animals that have died under human care or natural habitats in Japan. We also present a review of our cross-disciplinary strategy to conserve endangered species using germ cell preservation as a genetic resource with genomic and endocrine research.
To develop successful ex-situ breeding programs for the conservation of threatened wildlife, an understanding of reproductive endocrinology in the species of interest is essential. Fecal hormone metabolite patterns are widely used in this regard, but limited information is available regarding how these reflect blood hormone profiles. This study aimed to longitudinally monitor and compare circulating progestagen (sP) and estrogen (sE) concentrations, as well as fecal progestagen (fPM) and estrogen (fEM) metabolite concentrations with observed reproductive behaviors of African lionesses. For 18 months, blood sampling (n = 309) from five captive lionesses took place 1–7 times per week during positive reinforcement training. In parallel, over a period of 9 months, fecal samples (n = 129) from four of these lionesses were searched for daily and collected when found. Daily behavioral monitoring of all females enabled classification of reproductive stage according to presence/absence of characteristic behavioral events (i.e., “allowing mount”, “copulation”, “flirting run”, “lordosis”, “purring”, and “rolling”). Competitive enzyme immunoassays were used for steroid quantification. In total, 11 an-ovulatory and 16 ovulatory cycles (7 pregnancies and 9 pseudo-pregnancies) were assessed. When compared, the pattern of fecal hormone metabolite concentrations matched reproductive behavior more reliably than circulating steroid concentrations. Both sP and fPM patterns were correlated (n = 51; r = 0.86) and enabled identification of luteal phases, which helped to distinguish between pregnant and non-pregnant females. In contrast, detection of estrus by measurement of sE and fEM was not accurate regardless of the matrix evaluated.
The golden-headed lion tamarin (Leontopithecus chrysomelas) is an endangered primate endemic to the Atlantic Forest. Conservation efforts for the species involve applying reproductive biotechniques to preserve genetic resources and ensure the management of populations in both ex situ and in situ conditions. This study aims to initiate investigations into seminal and molecular factors influencing the reproductive potential of sexually mature males. Semen was collected using the penile vibrostimulation technique, and seminal parameters were assessed in two groups: the 'Old' group (average age 11.6 years; n=6) and the 'Young' group (average age 4.8 years; n=6). ANOVA results indicated age-related influences on plasma membrane integrity (p=0.049), acrosomal integrity (p=0.009), and DAB IV (p=0.026) for both groups. Linear regression revealed significant correlations between seminal parameters and age (plasma membrane integrity (p=0.021), acrosomal integrity (p=0.05), and DAB III (p=0.024)), alongside animal weight (plasma membrane integrity (p=0.010), acrosomal integrity (p=0.009), DAB III (p=0.33), and DAB IV (p=0.066)). In an effort to advance reproductive techniques and sperm selection, a protocol utilizing a discontinuous Percoll gradient was employed. Despite its effectiveness in isolating gametes, there were no significant gains in the reevaluated parameters post-selection, necessitating adjustments in the methodology. While semen cryopreservation is common in wild species, challenges arise due to seminal coagulum in many neotropical primate ejaculates, hindering gamete use in reproductive procedures. Given the precious nature of and the considerable effort involved in collecting semen from these animals, it would be desirable to maximize the sample's utility. The liquid fraction could be applied in reproductive biotechniques, while the spermatozoa contained in the clot could be utilized as a non-invasive approach for molecular evaluation of these gametes. This study established a protocol for RNA extraction from sperm retained in the seminal coagulum, highlighting its genetic richness often discarded post-processing. In summary, our study emphasizes the importance of early cryopreservation of semen to safeguard the reproductive potential of L. chrysomelas. Additionally, we propose further exploration of RNA quantity in gametes as a non-invasive tool for inferring male fertility, given the pivotal role of sperm RNA transcripts in regulating the activation of the female gamete and gene expression during early embryo development.

