Cryopreservation Cooling Rate Impacts Post-Thaw Sperm Motility and Survival in Litoria booroolongensis.

The cryopreservation and storage of gametes (biobanking) can provide a long-term, low-cost option for the preservation of population genetic diversity and is particularly impactful when applied to manage selective breeding within conservation breeding programs (CBPs). This study aimed to develop a sperm cryopreservation protocol for the critically endangered Booroolong frog (Litoria booroolongensis) to capture founder genetics within the recently established (est. 2019) CBP for this species. Hormone-induced sperm release was achieved using established protocols, and spermic urine samples were collected over a 6-h period. Pooled spermic urine samples (n = 3 males) were divided equally between two cryoprotectant (CPA) treatments and diluted by 1:5 (sperm:CPA) with either 15% (v/v) dimethyl sulfoxide + 1% (w/v) sucrose in simplified amphibian Ringer's (SAR; CPAA) or 10% (v/v) dimethylformamide + 10% (w/v) trehalose dihydrate in SAR (CPAB). The samples were cryopreserved in 0.25 mL straws using either a programmable freezer (FrA) or an adapted dry shipper method (FrB). The thawed samples were activated via dilution in water and assessed for viability and motility using both manual assessment and computer-assisted sperm analysis (CASA; 0 h, 0.5 h post-thaw). Upon activation, the survival and recovery of motility (total motility, forward progression and velocity) of cryopreserved sperm suspensions were higher for sperm preserved using FrB than FrA, regardless of CPA composition. This work supports our long-term goal to pioneer the integration of biobanked cryopreserved sperm with population genetic management to maximize restoration program outcomes for Australian amphibian species.

The Increasing Role of Short-Term Sperm Storage and Cryopreservation in Conserving Threatened Amphibian Species.

Multidisciplinary approaches to conserve threatened species are required to curb biodiversity loss. Globally, amphibians are facing the most severe declines of any vertebrate class. In response, conservation breeding programs have been established in a growing number of amphibian species as a safeguard against further extinction. One of the main challenges to the long-term success of conservation breeding programs is the maintenance of genetic diversity, which, if lost, poses threats to the viability and adaptive potential of at-risk populations. Integrating reproductive technologies into conservation breeding programs can greatly assist genetic management and facilitate genetic exchange between captive and wild populations, as well as reinvigorate genetic diversity from expired genotypes. The generation of offspring produced via assisted fertilisation using frozen-thawed sperm has been achieved in a small but growing number of amphibian species and is poised to be a valuable tool for the genetic management of many more threatened species globally. This review discusses the role of sperm storage in amphibian conservation, presents the state of current technologies for the short-term cold storage and cryopreservation of amphibian sperm, and discusses the generation of cryo-derived offspring.

Application of Reproductive Technologies to the Critically Endangered Baw Baw Frog, Philoria frosti.

Reproductive technologies (RTs) can assist integrated conservation breeding programs to attain propagation targets and manage genetic diversity more effectively. While the application of RTs to enhance the conservation management of threatened amphibians has lagged behind that of other taxonomic groups, a recent surge in research is narrowing the divide. The present study reports on the first application of RTs (hormone-induced spawning, hormone-induced sperm-release, and sperm cryopreservation) to the critically endangered Baw Baw frog, Philoria frosti. To determine the effect of hormone therapy on spawning success, male-female pairs were administered either 0 µg/g gonadotropin-releasing hormone agonist (GnRHa), 0.5 µg/g GnRHa, or 0.5 µg/g GnRHa + 10 µg/g metoclopramide (MET) (n = 6-7 pairs/treatment), and the number of pairs ovipositing, total eggs, and percent fertilisation success were quantified. To determine the effect of hormone therapy on sperm-release and to establish the peak time to collect sperm post-hormone administration, males were administered 0 IU/g (n = 4), or 20 IU/g hCG (n = 16). Total sperm, sperm concentration, and percent viability were quantified at 0, 2, 4, 6, 8, 10, and 12 h post-hormone administration. Overall, the percentage of pairs ovipositing was highest in the GnRHa + MET treatment, with 71% of pairs ovipositing, compared to 57% and 33% of pairs in the GnRHa and control treatments, respectively. The quantity of sperm released from males in response to hCG peaked at 4 h post-hormone administration, though it remained high up to 12 h. The percent sperm viability also peaked at 4 h post-administration (94.5%), exhibiting a steady decline thereafter, though viability remained above 77% throughout the 12 h collection period. The remaining sperm samples (n = 22) were cryopreserved using established protocols and biobanked for long-term storage and future conservation applications. The mean post-thaw sperm viability was 59%, and the percent total motility was 17%. The results from this preliminary study will direct further applications of RTs to the critically endangered Baw Baw frog to assist with species recovery.

Sperm cryopreservation in an Australian skink (Eulamprus quoyii).

Assisted reproductive technologies for population and genetic management for threatened herpetofauna have grown substantially in the past decade. Here we describe experiments to optimise sperm cryopreservation in a model squamate, the eastern water skink Eulamprus quoyii . Small, concentrated volumes of highly motile spermatozoa were reliably collected from adult male E. quoyii by non-lethal ventral massage. Samples were used to: (1) test whether protein-rich diluents, namely Beltsville poultry semen extender (BPSE) and TES and Tris (TEST) yolk buffer (TYB), improve post-thaw quality metrics compared with Dulbecco's phosphate-buffered saline (DPBS); and (2) compare the efficacy of these diluents in combination with either 1.35M glycerol or 1.35M dimethyl sulfoxide (DMSO) at two freezing rates, fast (approximately -20°C min-1 ) versus slow (-6°C min-1 ). Glycerol and DMSO performed equally well in preserving spermatozoa under slow freezing rates. Under these conditions, the use of the complex diluents BPSE and TYB significantly improved post-thaw total motility compared with DPBS. Complex interactions occurred between cryodiluent type, cryoprotectant and freezing rate when testing fast versus slow freezing rates among treatment groups. Under slow freezing rates, DMSO was better at preserving membrane integrity and motility, regardless of diluent type, but successful fast freezing required complex diluents to support motility and membrane integrity, which has implications for implementation in a field setting.

Follicle-stimulating hormone receptor (FSHR) alternative skipping of exon 2 or 3 affects ovarian response to FSH.

Genes critical for fertility are highly conserved in mammals. Interspecies DNA sequence variation, resulting in amino acid substitutions and post-transcriptional modifications, including alternative splicing, are a result of evolution and speciation. The mammalian follicle-stimulating hormone receptor (FSHR) gene encodes distinct species-specific forms by alternative splicing. Skipping of exon 2 of the human FSHR was reported in women of North American origin and correlated with low response to ovarian stimulation with exogenous follicle-stimulating hormone (FSH). To determine whether this variant correlated with low response in women of different genetic backgrounds, we performed a blinded retrospective observational study in a Turkish cohort. Ovarian response was determined as low, intermediate or high according to retrieved oocyte numbers after classifying patients in four age groups (<35, 35-37, 38-40, >40). Cumulus cells collected from 96 women undergoing IVF/ICSI following controlled ovarian hyperstimulation revealed four alternatively spliced FSHR products in seven patients (8%): exon 2 deletion in four patients; exon 3 and exons 2 + 3 deletion in one patient each, and a retention of an intron 1 fragment in one patient. In all others (92%) splicing was intact. Alternative skipping of exons 2, 3 or 2 + 3 were exclusive to low responders and was independent of the use of agonist or antagonist. Interestingly, skipping of exon 3 occurs naturally in the ovaries of domestic cats--a good comparative model for human fertility. We tested the signaling potential of human and cat variants after transfection in HEK293 cells and FSH stimulation. None of the splicing variants initiated cAMP signaling despite high FSH doses, unlike full-length proteins. These data substantiate the occurrence of FSHR exon skipping in a subgroup of low responders and suggest that species-specific regulation of FSHR splicing plays diverse roles in mammalian ovarian function.

Absence of seasonal changes in FSHR gene expression in the cat cumulus-oocyte complex in vivo and in vitro.

Domestic cat oocytes are seasonally sensitive to FSH. Compared with those collected during the breeding season, oocytes from the nonbreeding (NB) season require more FSH during in vitro maturation to achieve comparable developmental competence. This study tested the hypothesis that this seasonal variation was due to altered expression of FSH receptors (FSHR) and/or FSH-induced genes. Relative expression levels of FSHR mRNA and FSH-enhanced gene estrogen receptor ß (ESR2) were measured by qPCR in whole ovaries and immature cumulus-oocyte complexes (COCs) isolated from cat ovaries during the natural breeding vs NB seasons. Expression levels of FSH-induced genes prostaglandin-endoperoxide synthase 2 (PTGS2), early growth response protein-1 (EGR1), and epidermal growth factor receptor (EGFR) were examined in mature COCs from both seasons that were a) recovered in vivo or b) matured in vitro with conventional (1 µg/ml) or high (10 µg/ml) FSH concentrations. Overall, FSHR mRNA levels were lower in whole ovaries during the NB compared with breeding season but were similar in immature COCs, whereas ESR2 levels did not differ in either group between intervals. We observed changes in PTGS2, EGR1, and EGFR mRNA expression patterns across maturation in COCs within but not between the two seasons. The lack of seasonal differentiation in FSH-related genes was not consistent with the decreased developmental capacity of oocytes fertilized during the NB season. These findings reveal that the seasonal decrease in cat oocyte sensitivity to FSH occurs both in vivo and in vitro. Furthermore, this decline is unrelated to changes in expression of FSHR mRNA or mRNA of FSH-induced genes in COCs from antral follicles.

Stimulation of dopamine D2/D3 but not D1 receptors in the central amygdala decreases cocaine-seeking behavior.

Alterations in dopamine output within the various subnuclei of the amygdala have previously been implicated in cocaine reinforcement, as well as cocaine-seeking behavior. To elucidate the potential for increased stimulation of D1- and D2-like receptors (D1Rs and D2Rs, respectively) specifically in the central nucleus of the amygdala (CeA) to modulate cue- and cocaine-elicited reinstatement of cocaine-seeking behavior, we infused either the D1R agonist, SKF-38393 (0-4.0 microg/side) or the D2R agonist, 7-OH-DPAT (0-4.0 microg/side) into the CeA immediately prior to tests for cue and cocaine-primed reinstatement. We also examined the effects of 7-OH-DPAT on cocaine self-administration as a positive behavioral control. 7-OH-DPAT decreased cue-and cocaine-primed reinstatement, and reduced the number of cocaine infusions obtained during self-administration; SKF-38393 produced no discernable effects. The results suggest that enhanced stimulation of D2Rs, but not D1Rs, in the CeA is sufficient to inhibit expression of the incentive motivational effects of cocaine priming and cocaine-paired cues. Together with previous findings that D1R blockade attenuates reinstatement of cocaine-seeking behavior, the results suggest that D1R stimulation may be necessary, but not sufficient, to modulate the incentive motivational effects of cues and cocaine priming.

Effects of SCH-23390 infused into the amygdala or adjacent cortex and basal ganglia on cocaine seeking and self-administration in rats.

Amygdala D1 receptors have been implicated in the motivating effects of cocaine-conditioned cues and cocaine itself, but the specific nucleus involved is unclear. Thus, we infused the D1 antagonist, SCH-23390, into the rostral basolateral amygdala (rBLA), caudal basolateral amygdala (cBLA), or central amygdala (CEA), and tested its effects on self-administration of cocaine, as well as reinstatement of extinguished cocaine-seeking behavior by cocaine-conditioned cues or cocaine itself. Two anatomical controls, the posterior regions of basal ganglia (BG) and somatosensory/insular cortices (CTX), were also examined. Cocaine self-administration was increased and cue and cocaine reinstatement were decreased by SCH-23390 infusion into every region when examined across the hour test session, with the exception that cBLA infusion did not alter cocaine reinstatement. In the first 20 min of the session, when SCH-23390 was more localized in the target sites, self-administration was increased by infusion into the CEA, cBLA, BG, and CTX, with lesser increases in the rBLA. Cocaine reinstatement was attenuated during the first 20 min only by infusion into the CEA, rBLA, and CTX. Cue reinstatement was not reliably observed in the first 20 min, but there was a trend for attenuation by infusion into the cBLA, and surprisingly, significant attenuations in the BG and CTX. The findings suggest that D1 receptors in subregions of the amygdala play differential roles in the reinforcing/motivational effects of cocaine, while the cue reinstatement effects are less clear. Further research is needed to examine the novel findings that neighboring regions of the BG and CTX may play a role in motivation for cocaine.

Sex differences in spatial and non-spatial Y-maze performance after chronic stress.

Chronic restraint is known to alter hippocampal CA3 dendritic morphology and spatial memory in male rats. The present study examined whether female rats, which exhibit different anatomical adaptations to chronic stress than those of males, would also show spatial memory impairments. Male and female Sprague-Dawley rats were restrained for 6 h/day for 21 days, a time frame previously demonstrated to cause hippocampal CA3 dendritic atrophy. The day after the last restraint session, rats were tested on a Y-maze, a habituation task that can be used to assess spatial memory. Chronic stress impaired Y-maze performance in both sexes without affecting levels of locomotion as measured by total arm entries in the first minute. However, Y-maze performance of stressed females improved in 2-5 min when chronically stressed males continued to show poor Y-maze performance. The enhanced Y-maze performance of chronically stressed females occurred when total arm entries were higher compared to the entries made by males. Therefore, correlations were performed between total arm entries and spatial memory in 1 and 2-5 min. In the first minute when control females demonstrated functional spatial memory, female controls with the lowest locomotor levels exhibited the best performance. The correlations for stressed females were not significant, and neither were the correlations for any group in 2-5 min. Overall, these results show important sex differences in response to chronic stress with females exhibiting an ability to recover quickly from deficits in Y-maze performance.