Not just for males: Flehmen as a tool for detection of reproductive status and individual recognition across sexes in four African equid species.

Flehmen is frequently explained as part of male sexual behaviour, but it can also be associated with overmarking behaviour and thus individual recognition. We tested three explanatory hypotheses of flehmen behaviour: to detect sexual status of a female, to decide whether to overmark an individual, and to improve individual recognition. Additionally, we examined interspecific flehmen differences in the African equids. We observed 130 individuals of all 4 species among 15 groups in 5 zoos. We recorded 4445 eliminations: 142 were accompanied by flehmen and 1648 were inspected by another animal and followed by flehmen (n = 147 cases). As males of all age categories flehmened more often than females we conclude that flehmen serves to detect reproductive status of a female. However, this is not an exclusive explanation as animals of all sex and age categories flehmened when inspecting an elimination. Flehmen was not the predictor of overmarking. Nevertheless, we suggest that foals could use it for individual recognition. We found large interspecific differences with the highest rate of flehmen in African wild ass and least in mountain zebra. Thus, while the main function of flehmen is to detect female reproductive status, inter-individual and inter-species differences also play a role.

Why wait to mark? Possible reasons behind latency from olfactory exploration to overmarking in four African equid species.

Whereas most studies on overmarking in mammals analysed the rate of overmarking, that those investigate time between exploration of an olfactory stimulus and the response to it remain less common, with inconsistent results. We examined the latency in time between elimination by the sender and sniffing by the receiver, and from sniffing and overmarking, in four captive African equid species to explore differences among species, and among age and sex classes. We investigated these latency time periods in light of three potential hypotheses explaining overmarking behaviour in equids: social bonds, group cohesion, and intrasexual competition. Analysing 1684 events of sniffing and 719 of overmarking among 130 individuals, we found that (i) the time from elimination to overmarking was shorter among female friends and in parent-offspring dyads, proving support to the social bond hypothesis; (ii) intraspecific differences in time periods do not reflect the social organisation of species, thus not supporting the group cohesion hypothesis; (iii) males were more attracted to elimination of conspecifics than females, and female's eliminations were inspected longer, in line with the sexual competition hypothesis and/or reproductive behaviour. In addition, we found that the younger foals came to sniff eliminations faster than older ones, and in larger groups foals devoted longer time to sniffing the elimination before overmarking. We concluded that examination of the elimination could be driven by motivations other than the decision to overmark. Whereas overmarking serves to express bonds to a familiar individual, the latency of overmarking reflects more reproductive interests.

Changes in Phenotypes and DNA Methylation of In Vitro Aging Sperm in Common Carp Cyprinus carpio.

The purpose of the current study was to analyze phenotypic and functional characteristics of common carp (Cyprinus carpio) spermatozoa during in vitro aging and to investigate whether global DNA methylation is affected by sperm aging. Milt was collected from five individual males, stored in vitro on ice in a refrigerator for up to 96 h post stripping (HPS) and used to fertilize eggs with intervals of 1, 24 and 96 h. Computer-assisted sperm analysis and a S3e Cell Sorter was employed to determine the spermatozoa phenotypic characteristics (motility, velocity, concentration and viability). In addition, pH and osmolality of the seminal fluid and the capacity of the spermatozoa to fertilize, hatching rate and health of the resulting embryos were examined at different aging times. Whole-genome bisulfite sequencing was used to compare the global and gene-specific DNA methylation in fresh and aged spermatozoa. The results demonstrated that spermatozoa aging in common carp significantly affects their performance and thus the success of artificial fertilization. The methylation level at the cytosine-phosphate-guanine (CpG) sites increased significantly with 24 HPS spermatozoa compared to the fresh group at 1 HPS and then decreased significantly at 96 HPS. A more detailed investigation of gene specific differences in the DNA methylation was hindered by incomplete annotation of the C. carpio genome in the public databases.

Influence of Environmental Temperature and Hormonal Stimulation on the In Vitro Sperm Maturation in Sterlet Acipenser ruthenus in Advance of the Spawning Season.

Sturgeon sperm maturation occurs outside the testes during the transit of testicular spermatozoa (TS) through the kidneys and the Wolffian ducts. A method of in vitro TS maturation in sterlet Acipenser ruthenus was used to investigate the effects of temperature and hormonal stimulation of spermiation on the ability of TS to complete this process. Spermatozoa motility parameters after in vitro maturation of testicular sperm, concentrations of sex steroid hormones and testis morphology were studied in three groups of sterlet: (1) after overwintering in ponds (OW), (2) adapted to spawning temperature (ST), and (3) adapted to spawning temperature with hormonal induction of spermiation (ST-HI). Blood plasma concentrations of testosterone, 11-ketotestosterone and 17,20ß-dihydroxy-pregnenolone increased significantly after hormonal induction of spermiation (group ST-HI). In all groups, TS were not motile. After in vitro sperm maturation, motility was up to 60% only in group ST-HI. The data suggest that the ability of TS to be matured in vitro was not related to the environmental temperature, while hormonal stimulation of spermiation during the spawning season was an absolute requirement for optimal in vitro maturation.

Level of in vitro storage of the European catfish (Silurus glanis L.) eggs at different temperatures.

European catfish (Silurus glanis) is a commercially important freshwater fish originating from Eastern Europe. The objective of this study was to examine the short-term storage of its eggs especially in relation to maintaining a low level of malformation in newly hatched fry. The eggs from freshly spawned individuals were stored separately in cell incubators at 17 and 22 °C under aerobic conditions. Changes in fertilization, hatching, and malformation were examined in eggs stored at 1, 3, 5, and 7 h post-stripping. The sperm used for fertilization showed very good motility rates (84-90%) and curvilinear velocity (110-125 µm/s), and straight-line velocity did not drop below 77 µm/s. For all females, a temperature of 17 °C was better than 22 °C for egg storage in vitro. Egg fertilization and total hatching decreased rapidly after 7 h storage at 17 °C. The storage time of eggs in vitro to fertilization should therefore not exceed 5 h at 17 °C. In all females, there was no difference in the total number of eggs hatching between 1 and 3 h of egg storage at 17 °C. The storage time of eggs did not correlate with the level of malformations of the fry. However, the level of hatching and malformations was clearly affected by the storage temperature of eggs when it was > 17 °C. Analysis showed that the storage time of eggs, temperature of storage, and individual females had a significant influence on fertilization and total hatching rates. Regression analysis confirmed a low correlation of fertilization and hatching rates with storage time of eggs.

Improving motility and fertilization capacity of low-quality sperm of sterlet Acipenser ruthenus during storage.

Improvement of sperm quality with low motility by storage could ensure higher success of fertilization and maintain higher genetic diversity, especially for sturgeons, which as endangered species have limited broodstock and gametes. Sperm was collected from mature male sterlet Acipenser ruthenus and motility was evaluated using the CASA system; samples were categorized as GS 'good sperm' (>80%) or BS 'bad sperm' (<20%). Samples from both groups were incubated with seminal plasma from good- (GSP) and bad-quality sperm (BSP), respectively for 15 min, 6 h, 24 h and 96 h at 4 °C. Motility of BS incubated in GSP increased after different storage times compared to BS incubated in BSP, while the motility and velocity of GS incubated in BSP decreased compared to GS incubated in GSP. Fertilization rates were evaluated with samples stored for 15 min and 6 h post-stripping; fertilization and hatching rate of BS after incubation in GSP increased significantly compared to the BS incubated in BSP. Inorganic ion (Na+, K+, Cl-) concentrations and osmolality of BSP were significantly lower than that of GSP. These results indicated that sterlet sperm quality can be revitalized by incubation with GSP. Further, fertilization capacity of BS after incubation in GSP can reach similar levels to the good quality sperm (∼70%). Low ion concentration and osmolality in BSP may be a partial cause of low sperm quality. The current study is the first report on the capability to revitalize low quality sterlet sperm by storage in GSP.

Optimization of sterlet (Acipenser ruthenus) egg incubation.

Sterlet Acipenser ruthenus was used to assess egg and embryo development when incubated at 17 °C in Petri dishes placed in a hatchery tank (300 L recirculating dechlorinated water) with incubation occurring in a static tabletop system in an air-conditioned laboratory, or in a 700 L Q-cell incubator. Eggs in each dish were placed in a plastic box with 300 mL dechlorinated water. Separated eggs from three individual females were fertilized using pooled sperm from four males with there being four replicates. There were no differences (P > 0.05) in mean percentages of neurulation and embryos undergoing cleavage for eggs incubated in the hatchery tank and with use of the static tabletop system. Furthermore, there were no differences (P >  0.05) in percentage of embryos undergoing cleavage, neurulation and hatching for each female when eggs were incubated using the two systems. Results indicate a Petri dish placed in a small plastic box with 300 mL of dechlorinated water was adequate for incubation of sterlet eggs. Results of the study also indicate that with the static system: 1) eggs should be fertilized from each female to retain individual identity; 2) eggs should be dispersed in Petri dishes to avoid clumping; 3) water should be changed at 24 h, but not at 48 h (neurulation) post-fertilization; and 4) embryos that do not optimally develop should be removed the day after neurulation (72 h of post-fertilization period) and water should be exchanged every day subsequent to the 48 h time-point post-fertilization.

Effect of social organisation on interspecific differences in overmarking behaviour of foals in African equids.

Overmarking remains an unstudied topic in juvenile mammals. We have previously documented a very high rate of overmarking by foals in four captive African equid species: mountain zebra (Equus zebra), plains zebra (Equus quagga), Grévy's zebra (Equus grevyi), and African wild ass (Equus africanus). African equids vary interspecifically in their social organisation. Since differences in social organisation affect many mammalian behaviours, in this study we investigated interspecific differences in overmarking behaviour of foals, analysing only cases where elimination of any other individual was explored by a foal. We hypothesised that the pattern of overmarking by foals should reflect either differences in social organisation of the species or phylogenetic relations among them. We found that in all species very young foals explored mostly maternal eliminations, and this preference declined with increasing age of the foal and reflected the social organisation of the species; the highest overmarking rate was in species with high intragroup aggression (mountain zebra) and lowest in species with low intragroup aggression and which form crèches (African wild ass). Similarly, the rate of overmarking of the mother, as opposed to other herdmates, was associated with social organisation of the respective species. Thus, we found interspecific differences in overmarking by foals, which were associated with variability in social organisation. Since we also revealed differences between African wild ass and zebra behaviour in early stages of ontogeny, we cannot refute the effect of phylogeny on overmarking behaviour. Additionally, our results supported the identity sharing hypothesis as an explanation of overmarking.

Test of four hypotheses to explain the function of overmarking in foals of four equid species.

Overmarking occurs when one individual places its scent mark directly on top of the scent mark of another individual. Although it is almost ubiquitous among terrestrial mammals, we know little about the function of overmarking. In addition, almost all studies on mammalian overmarking behaviour dealt with adult individuals. Reports on this behaviour in juveniles are extremely rare, yet may elucidate the function of this behaviour. We tested four mutually non-exclusive hypotheses which might explain this behaviour in juveniles: (1) conceal the individual's scent identity, (2) announcement of association with other group members, especially the mother-i.e., sharing identity with the mother, (3) to prevent the next conception of the mother, i.e., parent-offspring conflict, and (4) an early expression of male sexual behaviour. We observed 43 foals (out of 108 individuals) from all African equid species (Equus africanus, E. grevyi, E. quagga, E. zebra) in five zoos. In total, we recorded 3340 eliminations; 260 of these events were overmarked by 38 individual foals representing all species. This represents one of the highest rates of overmarking ever recorded by mammalian juveniles. Foals of all species except African wild ass overmarked the mother more often than another herdmate: with male foals overmarked at a higher rate than female foals. Mothers preferred to overmark foals, but not exclusively their own foal. Our results provide support for the hypotheses that overmarking serves to share identity between foal and mother, and that it is an early expression of male sexual behaviour.

Effects of antifreeze proteins on cryopreserved sterlet (Acipenser ruthenus) sperm motility variables and fertilization capacity.

The effect of antifreeze proteins on sterlet, Acipenser ruthenus sperm motility variables and fertilization rate were investigated after cryopreservation. Two types of antifreeze proteins (AFPI or AFPIII) were used at concentrations of 0.1, 1, 10 and 100 µg/mL. The motility variables of fresh and cryopreserved sperm with and without addition of antifreeze proteins were evaluated by the Computer Assisted Semen Analyzer (CASA). The fertilization rate using about 200,000 spermatozoa per egg was evaluated after 54 h incubation at 17 °C during the early stage of organogenesis. The motility, curvilinear velocity and straight-line velocity of fresh sperm was 93 ± 5%, 128 ± 13 µm/s and 89 ± 9 µm/s, respectively. There was a significant decrease of sperm motility rate between fresh sperm and cryopreserved sperm with/without addition of antifreeze proteins. The greatest motility among thawed samples was in the sperm cryopreserved with 10 µg/mL of AFPI (56 ± 20%), however, these data were not different compared to the sperm without antifreeze proteins (49 ± 14%). No statistical variations were detected in curvilinear velocity nor straight-line velocity. The fertilization rate with fresh sperm was 67 ± 7%. No significant differences were detected in fertilization rate between fresh and cryopreserved spermatozoa with/without addition of antifreeze proteins, except the sperm cryopreserved with 100 µg/mL of AFPIII (39 ± 14%). Thus, it is concluded that addition of antifreeze proteins to cryopreservation medium do not improve nor have toxicity effects on the quality and fertilization capacity of sterlet sperm after thawing.