Record Longevity and Reproduction of an African Tick, Argas brumpti (Ixodida: Argasidae)

Argas brumpti Neumann is a large argasid (soft) tick that inhabits the drier areas of eastern and southern Africa. This species typically feeds on a wide variety of small to large mammals (including humans) and lizards, and resides in shallow caves, rocky areas, or dust-bath areas used by large mammals. Individuals of this species, collected as nymphs and adults from a semidesert area of Kenya and subsequently maintained under constant conditions in the laboratory, survived for 27 yr. Furthermore, after 8 yr of starvation and at least 4 yr after the last male died, at least one female laid eggs. The progeny developed into considerable numbers of both males and females, some of which are still living after 26 yr. The longevity of these ticks is apparently a record for any species of tick. The delay in reproduction likely represents long-term storage of viable sperm, also apparently a record for any species of tick.

Bicarbonate-Stimulated Membrane Reorganization in Stallion Spermatozoa

Classical in vitro fertilization (IVF) is still poorly successful in horses. This lack of success is thought to be due primarily to inadequate capacitation of stallion spermatozoa under in vitro conditions. In species in which IVF is successful, bicarbonate, calcium, and albumin are considered the key components that enable a gradual reorganization of the sperm plasma membrane that allows the spermatozoa to undergo an acrosome reaction and fertilize the oocyte. The aim of this work was to comprehensively examine contributors to stallion sperm capacitation by investigating bicarbonate-induced membrane remodelling steps, and elucidating the contribution of cAMP signalling to these events. In the presence of capacitating media containing bicarbonate, a significant increase in plasma membrane fluidity was readily detected using merocyanine 540 staining in the majority of viable spermatozoa within 15 min of bicarbonate exposure. Specific inhibition of soluble adenylyl cyclase (sAC) in the presence of bicarbonate by LRE1 significantly reduced the number of viable sperm with high membrane fluidity. This suggests a vital role for sAC-mediated cAMP production in the regulation of membrane fluidity. Cryo-electron tomography of viable cells with high membrane fluidity revealed a range of membrane remodelling intermediates, including destabilized membranes and zones with close apposition of the plasma membrane and the outer acrosomal membrane. However, lipidomic analysis of equivalent viable spermatozoa with high membrane fluidity demonstrated that this phenomenon was neither accompanied by a gross change in the phospholipid composition of stallion sperm membranes nor detectable sterol efflux (p > 0.05). After an early increase in membrane fluidity, a significant and cAMP-dependent increase in viable sperm with phosphatidylserine (PS), but not phosphatidylethanolamine (PE) exposure was noted. While the events observed partly resemble findings from the in vitro capacitation of sperm from other mammalian species, the lack of cholesterol removal appears to be an equine-specific phenomenon. This research will assist in the development of a defined medium for the capacitation of stallion sperm and will facilitate progress toward a functional IVF protocol for horse gametes.

DMPK hypermethylation in sperm cells of myotonic dystrophy type 1 patients

Myotonic dystrophy type 1 (DM1) is an autosomal dominant muscular dystrophy that results from a CTG expansion (50–4000 copies) in the 3′ UTR of the DMPK gene. The disease is classified into four or five somewhat overlapping forms, which incompletely correlate with expansion size in somatic cells of patients. With rare exception, it is affected mothers who transmit the congenital (CDM1) and most severe form of the disease. Why CDM1 is hardly ever transmitted by fathers remains unknown. One model to explain the almost exclusive transmission of CDM1 by affected mothers suggests a selection against hypermethylated large expansions in the germline of male patients. By assessing DNA methylation upstream to the CTG expansion in motile sperm cells of four DM1 patients, together with availability of human embryonic stem cell (hESCs) lines with paternally inherited hypermethylated expansions, we exclude the possibility that DMPK hypermethylation leads to selection against viable sperm cells (as indicated by motility) in DM1 patients.

Deep Learning-based Automated Rare Sperm Identification from Testes Biopsies

Non-obstructive azoospermia (NOA), the most severe form of male infertility, is currently treated using microsurgical sperm extraction (microTESE) to retrieve sperm cells for in vitro fertilization via intracytoplasmic sperm injection (IVF-ICSI). The success rate of this procedure for NOA patients is currently limited by the ability of andrologists to identify a few rare sperm cells among millions of background testis cells. To improve this success rate, we developed a convolution neural network (CNN) to detect rare sperm from low-resolution microscopy images of microTESE samples. Our CNN uses the U-Net architecture to perform pixel-based classification on image patches from brightfield microscopy, which is followed by morphological analysis to detect individual sperm instances. This CNN is trained using microscopy images of fluorescently labeled sperm, which is fixed to eliminate their motility, and doped into testis biopsies obtained from NOA patients. We initially tested this algorithm using purified sperm samples at different imaging magnifications in order to determine the upper bounds of performance. We then tested this algorithm by doping rare sperm cells into testis biopsy samples from NOA patients and found a sperm detection F1 score of 85.2%. These results demonstrate the potential to use automated microscopy to dramatically increase the amount of testis biopsy tissue that could be comprehensively examined, which greatly increases the chance of finding rare viable sperm, and thereby increases the success rates of IVF-ICSI for couples with NOA.

PSXV-13 The activity of thiol cathepsins in seminal plasma of stallions with different percentages of viable sperm after freezing-thawing

The aim of this work was to study the activity of cathepsins B, L and H in the seminal plasma of stallions with normal (>25%, n = 11) and low (< 25%, n = 13) percentage of viable sperm after freezing-thawing. Sperm of 24 Arabian stallions aged from 5 to 20 years (12.1 ± 4.8 years on average) was collected during the breeding season (February-May). The activity of cathepsins in spermoplasm was studied by the spectrofluorometric method (System 3 Scanning Spectrofluorometry, Optical technology devices, inc. Elmstord, New York, 10523) by Barrett&Kirschke. The viability of the sperm was determined after its freezing-thawing. Sperm smears were eosin stained and live:dead-ratio was examined using an Olympus BX41 phase contrast microscope (Olympus Corporation, Japan). The significance of differences in the studied groups was determined using the Mann-Whitney U-test. There were no significant differences in the activity of catepsins B and L in the spermoplasm of stallions with normal and low percentages of viable spermatozoa after freezing-thawing. It was found that in the group of stallions with a normal percentage of viable spermatozoa after freezing-thawing, the activity of cathepsin H in the spermoplasm was significantly higher than in the group with a low percentage of viable spermatozoa (P = 0.0219). Free radicals formed during freezing-thawing of sperm can damage cell membranes, leading to loss of sperm viability. Thiol cathepsins are involved in the degradation of oxidatively modified proteins and, apparently, it is cathepsin H that is most actively involved in this process in the sperm of stallions. We assume that the low activity of cathepsin H in the seminal plasma of stallions with a low percentage of viable spermatozoa indicates a high involvement of this enzyme in the protection of sperm membranes from oxidative damage. The research was supported by the Russian Science Foundation grant No. 20-16-00101.

476 Effect of Holding Period on Viability, Acrosome Status and Intracellular Zinc of Boar Sperm During Liquid Storage

Sperm capacitation results in acrosomal remodeling, increased membrane fluidity, plasma membrane fusability and hyperactivated sperm. Distinct sperm zinc signatures reflect intracellular zinc content, capacitation status, and viability. Therefore, flow cytometric assessment could be a useful tool to evaluate sperm quality and longevity during liquid storage of boar semen. Cooled semen (17°C) from commercial boars (n = 12) were assessed at Day-1, -4 and -7 after collection for motility (IVOS II, Hamilton Thorne, Beverly, MA), intracellular zinc and viability. Samples were stained with 8 µg/mL Hoecsht-33342 (Hoe), 1 µM FluoZinTM-3-AM (Zinc), 10 µM propidium iodide (PI) and 5.6 µg/mL PNA-Alexa647TM (Molecular Probes, Eugene, OR), incubated for 30 min, washed with PBS, and aditionally incubated for 30 min. Acquisition and analysis of 20000 events were performed using Bio-Rad ZE5 Cell Analyzer (Hercules, CA) and FLowJoTM (Ashland, OR). Hoe+ events were gated into Zinc/PI plot to determine viable sperm with high Zinc (Zinc+/PI-). Likewise, Hoe+ events were gated into PI/PNA plot and then into Zinc histogram to detect viable-intact-acrosome with high Zinc (Zn+/PI-/PNA-). Data were analyzed by mixed model for repeated measures, Tukey-adjusted pairwise comparisons and Pearson’s correlation. Viable sperm at Day-1 (86.0±3.1; emmean±SM) and Day-4 (84.7±2.8) showed similar percentage of Zinc+ sperm, and both significantly higher compared to Day-7 (52.2±2.8). Also, viable-intact-acrosome sperm displayed similar percentage of Zinc+ sperm at Day-1 (93.8±3.4) and Day-4 (91.6±3.4) and both higher than sperm at Day-7 (64.5±3.1; P < 0.0001). Percentages of Zinc+ sperm were higher (P < 0.001) when viable sperm had intact acrosome within Day. Percentages of Zinc+ sperm strongly correlated to viable-intact-acrosome sperm (r=0.76, P < 0.0001) and total motility (r=0.65, P < 0.0001). We conclude that sperm having intact plasmatic membrane and acrosome display higher intracellular zinc content which is related to capacitated status, remaining non-capacitated until at least to Day-4 after collection in the tested extender.

Effect of adding anethole to the cryopreservation medium (powdered coconut water) on morphology, kinetics, and oxidative stress of buck sperm

The quality of post-thawing goat sperm is critical to the success of artificial insemination protocols and may be influenced by extenders, cryoprotectants, and antioxidant substances. Therefore, the objective of this study was to evaluate the effects of the antioxidant anethole on goat sperm diluted in preservation medium based on powdered coconut water (ACP-101c) and frozen. For that, each ejaculate was submitted to the following treatments: ACP-101c (control); control plus supplementation with 30, 300, or 2000 μg/mL anethole. The samples were thawed and evaluated for morphology, kinetics, membrane integrity, and reactive oxygen species (ROS). The addition of anethole increased morphological abnormalities (P < 0.05), however, it did not affect sperm kinetics. Flow cytometry analysis showed that sperm cells cryopreserved with 300 μg/mL anethole had lower acrosome integrity than those cryopreserved in other treatments. Evaluation of oxidative stress revealed that cells stored in the presence of 2000 μg/mL anethole had small amounts of ROS when compared to those preserved in the control medium alone or supplemented with 300 μg/mL anethole (P < 0.05). After cryopreservation of sperm with 2000 μg/mL anethole, the highest percentage of viable sperm without ROS was observed (P < 0.05). In conclusion, despite reducing ROS levels, the supplementation of anethole in ACP-101c did not affect sperm kinetics or membrane integrity post-thawing, however, it did cause morphological damage to sperm.

Reproductive Anatomy of Chondrichthyans: Notes on Specimen Handling and Sperm Extraction. II. Sharks and Chimaeras

The chondrichthyan fishes, which comprise sharks, rays, and chimaeras, are one of the most threatened groups of vertebrates on the planet. Given this situation, an additional strategy for the protection of these species could be the ex situ conservation projects developed in public aquaria and research centers. Nevertheless, to increase sustainability and to develop properly in situ reintroduction strategies, captive breeding techniques, such as sperm extraction and artificial insemination, should be developed. These techniques are commonly used in other threatened species and could be also used in chondrichthyans. However, the different reproductive morphologies found in this group can complicate both processes. Therefore, a comparison of the reproductive anatomy of eight distinct chondrichthyans, with an emphasis on those important differences when performing sperm extraction or artificial insemination, is carried out herein. Sharks and chimaeras belonging to the Scyliorhinidae, Carcharhinidae, Centrophoridae, Etmopteridae, Hexanchidae, and Chimaeridae families were obtained from commercial fisheries, public aquaria, and stranding events. In addition, the process of obtaining viable sperm samples through cannulation, abdominal massage, and oviducal gland extraction is described in detail for both living and dead animals.

Initial collection, characterization, and storage of tuatara (Sphenodon punctatus) sperm offers insight into their unique reproductive system

Successful reproduction is critical to the persistence of at-risk species; however, reproductive characteristics are understudied in many wild species. New Zealand’s endemic tuatara (Sphenodon punctatus), the sole surviving member of the reptile order Rhynchocephalia, is restricted to 10% of its historic range. To complement ongoing conservation efforts, we collected and characterized mature sperm from male tuatara for the first time. Semen collected both during mating and from urine after courting contained motile sperm and had the potential for a very high percentage of viable sperm cells (98%). Scanning electron microscopy revealed a filiform sperm cell with distinct divisions: head, midpiece, tail, and reduced end piece. Finally, our initial curvilinear velocity estimates for tuatara sperm are 2–4 times faster than any previously studied reptile. Further work is needed to examine these trends at a larger scale; however, this research provides valuable information regarding reproduction in this basal reptile.