Optimization of a non-activating medium for short-term chilled storage of barramundi (Lates calcarifer) testicular spermatozoa.

Reliable short-term chilled sperm storage is a critical prerequisite to using advanced reproductive techniques for captive breeding of barramundi (Asian sea bass; Lates calcarifer). Marine Ringer's solution (MRS) is a common non-activating medium (NAM) and has previously been used to store sperm from wild-caught barramundi. However, MRS-stored spermatozoa from captive-bred barramundi were observed to lyse within 30 min incubation. Therefore, this study aimed to optimize the composition of NAM for short-term chilled storage by characterizing and mimicking the biochemical profile of seminal and blood plasma of captive-bred barramundi. To further understand the effect of each component, osmolality was first examined to determine its effect on sperm viability. Thereafter, the effects of NaHCO3, pH, and Na+ and K+ concentrations on sperm motility were investigated. Optimization of the NAM formula was achieved through iterative adaptions. The increase in NAM osmolality from 260 to 400 mOsm/kg led to a significant improvement in sperm viability. Moreover, using HEPES instead of NaHCO3 as buffering agent significantly enhanced sperm motility and velocity. As a result, sperm samples diluted with optimized NAM (185 mM NaCl, 5.1 mM KCl, 1.6 mM CaCl2·2H2O, 1.1 mM MgSO4·7H2O, 10.0 mM HEPES, 5.6 mM D+ glucose, 400 mOsm/kg, pH 7.4) and stored at 4 °C showed no significant loss in total motility for up to 48 h and retained progressive motility for up to 72 h. The optimized NAM developed in this study significantly extended the functional lifespan of spermatozoa during chilled storage, permitting the ongoing development of advanced reproductive technologies for barramundi.

Appeasing Pheromones for the Management of Stress and Aggression during Conservation of Wild Canids: Could the Solution Be Right under Our Nose?

Thirty-six species of canid exist globally, two are classified as critically endangered, three as endangered, and five as near threatened. Human expansion and the coinciding habitat fragmentation necessitate conservation interventions to mitigate concurrent population deterioration. The current conservation management of wild canids includes animal translocation and artificial pack formation. These actions often cause chronic stress, leading to increased aggression and the suppression of the immune and reproductive systems. Castration and pharmaceutical treatments are currently used to reduce stress and aggression in domestic and captive canids. The undesirable side effects make such treatments inadvisable during conservation management of wild canids. Pheromones are naturally occurring chemical messages that modulate behaviour between conspecifics; as such, they offer a natural alternative for behaviour modification. Animals are able to distinguish between pheromones of closely related species through small compositional differences but are more likely to have greater responses to pheromones from individuals of the same species. Appeasing pheromones have been found to reduce stress- and aggression-related behaviours in domestic species, including dogs. Preliminary evidence suggests that dog appeasing pheromones (DAP) may be effective in wild canids. However, the identification and testing of species-specific derivatives could produce more pronounced and beneficial behavioural and physiological changes in target species. In turn, this could provide a valuable tool to improve the conservation management of many endangered wild canids.

Susceptibility of boar spermatozoa to heat stress using in vivo and in vitro experimental models.

Induction of heat stress as an experimental procedure in animals is commonly used to examine heat-related impacts on sperm quality. This study aimed to develop potential heat stress models that could be used at any time of the year, to advance the study of seasonal infertility in the pig under controlled conditions. Heat stress was induced by either housing boars (n = 6) at 30 °C inside a hot room for 42 days (55-65% humidity; LD 12:12 h; in vivo), or by heating boar semen (n = 7) for 30 min at various temperatures (35.5, 38.8, 40, 42, 46, 50, 54 and 60 °C; in vitro). Sperm motility was then characterized by computer-assisted sperm analysis (CASA; IVOS version 10: Hamilton Thorne, USA), and DNA integrity was evaluated by terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) and flow cytometry. Our in vivo hot room model induced biologically meaningful levels of DNA damage in boar spermatozoa (10.1 ± 1.9 hot room vs. 6.7 ± 1.7% control; P > 0.05), although not statistically significant from controls. Moreover, sperm concentration and motility parameters did not differ between treatments (P > 0.05). Compared to the 38.8 °C control, our in vitro heat shock model significantly increased sperm DNA damage after incubation at 54 and 60 °C (3.0 ± 1.0, 2.9 ± 1.0, 1.2 ± 0.3, 2.5 ± 0.7, 9.0 ± 3.7, 16.2 ± 7.1, 14.2 ± 5.8 and 41.8 ± 18.6% respectively; P ≤ 0.05). However, these temperatures rendered sperm completely immotile or dead, with most motility parameters declining rapidly to zero above 40 or 42 °C. In conclusion, our results suggest that temperature combined with individual factors may contribute to a boar's overall susceptibility to heat stress. Refinement of these models particularly of the in vitro heat shock model could be further pursued to overcome environmental variability, reduce whole animal experiments and provide a putative diagnostic fertility screening tool to evaluate heat tolerance in the boar.

Antioxidant supplementation mitigates DNA damage in boar (Sus scrofa domesticus) spermatozoa induced by tropical summer.

Heat stress-induced sperm DNA damage has recently been demonstrated in boars during tropical summer; which could negatively impact early embryo survival and litter size in sows. Given the boar's inefficient capacity to sweat, non-pendulous scrotum and low antioxidant activity in seminal plasma, elevated endogenous levels of antioxidants are needed to combat reactive oxygen species induced during periods of heat stress. This should prevent the build-up of pathological levels of DNA damage in boar spermatozoa. Our aim was to investigate whether a combined antioxidant supplement could mitigate sperm DNA damage in boars exposed to tropical summer conditions. Terminal deoxynucleotidyl transferase dUTP nick end labelling and flow cytometry of 20,000 spermatozoa/boar/treatment revealed that boar diets supplemented with 100 g/day custom-mixed antioxidant during peak wet summer effectively reduced sperm DNA damage by as much as 55% after 42 and 84 days treatment respectively (16.1 ± 4.9 peak wet control vs. 9.9 ± 4.5 42 day vs. 7.2 ± 1.6% 84 day treatments; P ≤ 0.05). Supplementation did not improve sperm concentration beyond control levels for either season (P > 0.05); nor alter total motility, progressive motility or several other motion parameters measured by computer assisted sperm analysis of 20 x 106 sperm/mL at 38°C (P > 0.05). Antioxidant supplementation during tropical summer appears to mitigate the negative impact of heat stress on DNA integrity but not concentration nor motility of boar spermatozoa; which may provide one solution to the problem of summer infertility in the pig.

Dog appeasing pheromone prevents the androgen surge and may reduce contact dominance and active submission after stressful interventions in African wild dogs (Lycaon pictus).

The endangered African wild dog (AWD; Lycaon pictus) is a highly social canid living in packs with a separate male and female hierarchy. Immobilisation, handling and translocations are acute stressors for AWDs, however such interventions are often needed for species management. In addition, new pack formation or temporary pack separation can lead to an increase in intra-pack aggression. The goal of this double-blinded placebo-controlled study conducted in captive zoo populations was to evaluate whether dog appeasing pheromone (DAP) reduces behavioural stress and faecal glucocorticoid metabolite levels (fGCM) normally associated with pack separation, immobilisation and reintroduction (SIR), and to assess whether this reduces aggressive behaviours and faecal androgen metabolite levels (fAM). Four packs (n = 11 males) were treated with DAP and 4 packs (n = 12 males) were treated with a placebo solution, applied at the end of anaesthesia. Behavioural interactions as well as fGCM and fAM were determined from 3 days before until 4-6 days after SIR. No effect of DAP on fGCM was observed, however, fAM increased after SIR in placebo but not DAP treated animals. Moreover, on the day of reintroduction, DAP treated packs tended to have lower rates of contact-dominance and active-submission behaviour, but higher rates of non-contact dominance behaviour. As these effects could decrease the risk of agonistic interactions, DAP may be a useful tool to help manage new pack formations and temporary pack separation.

Social rank does not affect sperm quality in male African wild dogs (Lycaon pictus).

Sperm banking and AI could benefit endangered African wild dog conservation. However, it is unclear whether their dominance hierarchy causes a decrease in reproductive and sperm quality parameters in subordinate males that typically do not breed. In this study, we investigated the effect of social rank on male reproductive parameters, including faecal androgen and glucocorticoid metabolite concentrations, prostate and testes volume, preputial gland size, semen collection success and sperm quality. Samples were obtained from captive males (prebreeding season: n=12 from four packs; breeding season: n=24 from seven packs) that were classified as alpha (dominant), beta or gamma (subordinates) based on the frequency of dominant versus submissive behaviours. In the prebreeding season, semen was successfully collected from all alpha but only half the subordinate males, with urine contamination (associated with lower rank) significantly reducing total and progressive motility, sperm motility index, normal sperm morphology and acrosome integrity. The breeding season was associated with a significant increase in faecal androgens, prostate and testis volume, as well as progressive motility and the total number of spermatozoa ejaculated. However, with the exception of prostate volume (mean±s.e.m: 12.5±4.5, 7.1±1.0 and 7.3±1.0cm3 in alpha, beta and gamma males respectively; P=0.035), all other reproductive and sperm quality parameters did not differ between males of each social rank. In conclusion, reproductive suppression of subordinate males appears to be behaviourally mediated, because males of all social ranks produce semen of similar quality, making them suitable candidates for sperm banking, particularly during the breeding season when sperm quality improves.

Mitochondrial DNA replication is initiated at blastocyst formation in equine embryos.

Intracytoplasmic sperm injection is the technique of choice for equine IVF and, in a research setting, 18-36% of injected oocytes develop to blastocysts. However, blastocyst development in clinical programs is lower, presumably due to a combination of variable oocyte quality (e.g. from old mares), suboptimal culture conditions and marginal fertility of some stallions. Furthermore, mitochondrial constitution appears to be critical to developmental competence, and both maternal aging and invitro embryo production (IVEP) negatively affect mitochondrial number and function in murine and bovine embryos. The present study examined the onset of mitochondrial (mt) DNA replication in equine embryos and investigated whether IVEP affects the timing of this important event, or the expression of genes required for mtDNA replication (i.e. mitochondrial transcription factor (TFAM), mtDNA polymerase γ subunit B (mtPOLB) and single-stranded DNA binding protein (SSB)). We also investigated whether developmental arrest was associated with low mtDNA copy number. mtDNA copy number increased (P<0.01) between the early and expanded blastocyst stages both invivo and invitro, whereas the mtDNA:total DNA ratio was higher in invitro-produced embryos (P=0.041). Mitochondrial replication was preceded by an increase in TFAM but, unexpectedly, not mtPOLB or SSB expression. There was no association between embryonic arrest and lower mtDNA copy numbers.

Tropical summer induces DNA fragmentation in boar spermatozoa: implications for evaluating seasonal infertility.

Summer infertility continues to undermine pig productivity, costing the pig industry millions in annual losses. The boar's inefficient capacity to sweat, non-pendulous scrotum and the extensive use of European breeds in tropical conditions, can make the boar particularly vulnerable to the effects of heat stress; however, the link between summer heat stress and boar sperm DNA damage has not yet been demonstrated. Semen from five Large White boars was collected and evaluated during the early dry, late dry and peak wet seasons to determine the effect of seasonal heat stress on the quality and DNA integrity of boar spermatozoa. DNA damage in spermatozoa during the peak wet was 16-fold greater than during the early dry and nearly 9-fold greater than during the late dry season. Sperm concentration was 1.6-fold lower in the peak wet than early dry whereas no difference was found across several motility parameters as determined by computer-assisted sperm analysis. These results demonstrate that tropical summer (peak wet season) induces DNA damage and reduces concentration without depressing motility in boar spermatozoa, suggesting that traditional methods of evaluating sperm motility may not detect inherently compromised spermatozoa. Boar management strategies (such as antioxidant supplementation) need to be developed to specifically mitigate this problem.

Maternal age and in vitro culture affect mitochondrial number and function in equine oocytes and embryos.

Advanced maternal age and in vitro embryo production (IVP) predispose to pregnancy loss in horses. We investigated whether mare age and IVP were associated with alterations in mitochondrial (mt) DNA copy number or function that could compromise oocyte and embryo development. Effects of mare age (<12 vs ≥12 years) on mtDNA copy number, ATP content and expression of genes involved in mitochondrial replication (mitochondrial transcription factor (TFAM), mtDNA polymerase γ subunit B (mtPOLB) and mitochondrial single-stranded DNA-binding protein (SSB)), energy production (ATP synthase-coupling factor 6, mitochondrial-like (ATP-synth_F6)) and oxygen free radical scavenging (glutathione peroxidase 3 (GPX3)) were investigated in oocytes before and after in vitro maturation (IVM), and in early embryos. Expression of TFAM, mtPOLB and ATP-synth-F6 declined after IVM (P<0.05). However, maternal age did not affect oocyte ATP content or expression of genes involved in mitochondrial replication or function. Day 7 embryos from mares ≥12 years had fewer mtDNA copies (P=0.01) and lower mtDNA:total DNA ratios (P<0.01) than embryos from younger mares, indicating an effect not simply due to lower cell number. Day 8 IVP embryos had similar mtDNA copy numbers to Day 7 in vivo embryos, but higher mtPOLB (P=0.013) and a tendency to reduced GPX3 expression (P=0.09). The lower mtDNA number in embryos from older mares may compromise development, but could be an effect rather than cause of developmental retardation. The general down-regulation of genes involved in mitochondrial replication and function after IVM may compromise resulting embryos.

Oviduct binding ability of porcine spermatozoa develops in the epididymis and can be advanced by incubation with caudal fluid.

The sperm reservoir is formed when spermatozoa bind to the epithelium of the uterotubal junction and caudal isthmus of the oviduct. It is an important mechanism that helps synchronize the meeting of gametes by regulating untimely capacitation and polyspermic fertilization. This study investigated the influence of epididymal maturation and caudal fluid on the ability of spermatozoa to bind to oviduct epithelium using a model porcine oviduct explant assay. Spermatozoa from the rete testis, middle caput (E2-E3), middle corpus (E6), and cauda (E8) of Large White or Large White × Landrace boars aged 10 to 14 months were diluted in modified Androhep solution and incubated with porcine oviduct explants. Results reported in this study support our hypothesis that testicular spermatozoa need to pass through the regions of the epididymis to acquire the ability to bind to the oviduct. There was a sequential increase in the number of spermatozoa that bound to oviduct explants from the rete testis to caudal epididymis. Binding of caudal spermatozoa to isthmic explants was the highest (15.0 ± 1.2 spermatozoa per 1.25 mm(2), mean ± standard error of the mean; P ≤ 0.05) and lowest by spermatozoa from the rete testis (2.0 ± 0.3 per 1.25 mm(2)), and higher to isthmus from sows compared to gilts (35.8 ± 6.7 per 1.25 mm(2) vs. 14.8 ± 3.0 per 1.25 mm(2); P ≤ 0.05). Binding of ejaculated spermatozoa to porcine isthmus was higher than that for caudal spermatozoa (26.3 ± 1.4 per 1.25 mm(2) vs. 15.0 ± 0.8 per 1.25 mm(2); P ≤ 0.05) and higher to porcine than to bovine isthmus (26.3 ± 2.3 per 1.25 mm(2) vs. 18.8 ± 1.9 per 1.25 mm(2); P ≤ 0.05). Incubation of spermatozoa from the caput and corpus in caudal fluid increased the ability of spermatozoa to bind to the oviduct epithelium (P ≤ 0.05). In conclusion, the capacity of testicular spermatozoa to bind to the oviduct epithelium increases during their maturation in the epididymis and can be advanced by components of the caudal fluid.

Establishing reference genes for use in real-time quantitative PCR analysis of early equine embryos.

Real-time quantitative PCR (qPCR) is invaluable for investigating changes in gene expression during early development, since it can be performed on the limited quantities of mRNA contained in individual embryos. However, the reliability of this method depends on the use of validated stably expressed reference genes for accurate data normalisation. The aim of the present study was to identify and validate a set of reference genes suitable for studying gene expression during equine embryo development. The stable expression of four carefully selected reference genes and one developmentally regulated gene was examined by qPCR in equine in vivo embryos from morula to expanded blastocyst stage. SRP14, RPL4 and PGK1 were identified by geNorm analysis as stably expressed reference genes suitable for data normalisation. RPL13A expression was less stable and changed significantly during the period of development examined, rendering it unsuitable as a reference gene. As anticipated, CDX2 expression increased significantly during embryo development, supporting its possible role in trophectoderm specification in the horse. In summary, it was demonstrated that evidence-based selection of potential reference genes can reduce the number needed to validate stable expression in an experimental system; this is particularly useful when dealing with tissues that yield small amounts of mRNA. SRP14, RPL4 and PGK1 are stable reference genes suitable for normalising expression for genes of interest during in vivo morula to expanded blastocyst development of horse embryos.

Artificial insemination in marsupials.

Assisted breeding technology (ART), including artificial insemination (AI), has the potential to advance the conservation and welfare of marsupials. Many of the challenges facing AI and ART for marsupials are shared with other wild species. However, the marsupial mode of reproduction and development also poses unique challenges and opportunities. For the vast majority of marsupials, there is a dearth of knowledge regarding basic reproductive biology to guide an AI strategy. For threatened or endangered species, only the most basic reproductive information is available in most cases, if at all. Artificial insemination has been used to produce viable young in two marsupial species, the koala and tammar wallaby. However, in these species the timing of ovulation can be predicted with considerably more confidence than in any other marsupial. In a limited number of other marsupials, such precise timing of ovulation has only been achieved using hormonal treatment leading to conception but not live young. A unique marsupial ART strategy which has been shown to have promise is cross-fostering; the transfer of pouch young of a threatened species to the pouches of foster mothers of a common related species as a means to increase productivity. For the foreseeable future, except for a few highly iconic or well studied species, there is unlikely to be sufficient reproductive information on which to base AI. However, if more generic approaches can be developed; such as ICSI (to generate embryos) and female synchronization (to provide oocyte donors or embryo recipients), then the prospects for broader application of AI/ART to marsupials are promising.

A perspective on the role of emerging technologies for the propagation of companion animals, non-domestic and endangered species.

Assisted reproductive technologies (ART) have been used successfully in humans, domestic and laboratory species for many years. In contrast, our limited knowledge of basic reproductive physiology has restricted the application of ART in companion animal, non-domestic and endangered species (CANDES). Although there are numerous benefits, and in some cases a necessity, for applying ART for the reproductive and genetic management of CANDES, the challenges encountered with even the most basic procedures have limited the rate of progress. In this foreword we discuss the status of conventional ART, such as artificial insemination and in vitro fertilisation, as well as their benefits and inherent difficulties when applied to CANDES. It is upon these techniques, and ultimately our knowledge of basic reproductive physiology, that the success of emerging technologies, such as those described in this special issue, are dependent for success.

Birth of pouch young after artificial insemination in the tammar wallaby (Macropus eugenii).

Timing of artificial insemination (AI) in marsupials is critical because fertilization must occur before mucin coats the oocyte during passage through the oviduct. In this study, timing and the site of insemination were examined to develop AI in the tammar wallaby (Macropus eugenii). Birth and postpartum (p.p.) estrus was synchronized in 46 females. Epididymal spermatozoa (n=4) or semen collected by electroejaculation (n=42) were inseminated early (4-21 h p.p.) into the urogenital sinus (n=7), the anterior vaginal culs de sac (n=7), the uterus by transcervical catheter (n=5), or the uterus by injection (intrauterine artificial insemination, IUAI) (n=5). A further 16 females were inseminated late (19-48 h p.p.) by IUAI. All females were monitored for birth. A third group of six females was inseminated late (21-54 h p.p.) by IUAI and 0.4-6.6 h later, sperm had reached the oviduct in all animals. In total, an oocyte to which spermatozoa were attached was recovered and two young were born after IUAI using epididymal (n=1) or electroejaculated (n=2) spermatozoa, but no young resulted from insemination at other sites. Two females were successfully inseminated at 43 and 47 h p.p., later than most other animals, and the third was inseminated much earlier (18 h p.p.) but with highly motile spermatozoa. These young represent the first macropodids born by AI and the first marsupials conceived using epididymal spermatozoa.

Sperm transport, size of the seminal plug and the timing of ovulation after natural mating in the female tammar wallaby Macropus eugenii.

The distribution of spermatozoa and seminal plug in the reproductive tract and the timing of ovulation were examined at various times in a naturally mated monovular macropodid marsupial, namely the tammar wallaby (Macropus eugenii). After the first post partum (p.p.) mating, 28 females were isolated and their reproductive tracts dissected at 0.5, 6, 18, 36 and 40 h post coitum (p.c.). Each tract was ligated into 13 major anatomical sections and spermatozoa and eggs were recovered by flushing. Mating was possibly delayed by handling and occurred 21.7 +/- 2.5 h p.p. in these animals. Copulation lasted 7.8 +/- 0.7 min. Within 0.5 h after a single mating, the tract contained 25.8 +/- 10.2 x 10(6) spermatozoa and 21.6 +/- 8.8 g of seminal plug, 96% and 70% of which was lost within 6 h p.c. respectively. Spermatozoa reached the uterus, isthmus and ampulla of the oviduct on the side of the developing follicle within 0.5, 6 and 18 h p.c., respectively, and a uterine population of 26.1 +/- 12.10(3) spermatozoa was maintained for over 40 h. Sperm numbers were reduced at the cervix (up to 57-fold) and uterotubule junction (eight-fold) and only one in approximately 7500 ejaculated spermatozoa (3.4 +/- 0.9 x 10(3)) reached the oviduct on the follicle side. Differential transport of spermatozoa was not observed. Although the numbers of spermatozoa were reduced in the parturient uterus, they were highly variable and were not significantly different to those in the non-parturient uterus. Ovulation and recovery of sperm-covered eggs from the isthmus occurred 36-41 h p.c. (49-72 h p.p.). In contrast with the polyovular dasyurid and didelphid marsupials, the tammar wallaby ejaculates large numbers of spermatozoa, but transport is relatively inefficient and sperm storage in the tract before ovulation is limited.