The evolutionary maintenance of ancient recombining sex chromosomes in the ostrich.

Sex chromosomes have evolved repeatedly across the tree of life and often exhibit extreme size dimorphism due to genetic degeneration of the sex-limited chromosome (e.g. the W chromosome of some birds and Y chromosome of mammals). However, in some lineages, ancient sex-limited chromosomes have escaped degeneration. Here, we study the evolutionary maintenance of sex chromosomes in the ostrich (Struthio camelus), where the W remains 65% the size of the Z chromosome, despite being more than 100 million years old. Using genome-wide resequencing data, we show that the population scaled recombination rate of the pseudoautosomal region (PAR) is higher than similar sized autosomes and is correlated with pedigree-based recombination rate in the heterogametic females, but not homogametic males. Genetic variation within the sex-linked region (SLR) (π = 0.001) was significantly lower than in the PAR, consistent with recombination cessation. Conversely, genetic variation across the PAR (π = 0.0016) was similar to that of autosomes and dependent on local recombination rates, GC content and to a lesser extent, gene density. In particular, the region close to the SLR was as genetically diverse as autosomes, likely due to high recombination rates around the PAR boundary restricting genetic linkage with the SLR to only ~50Kb. The potential for alleles with antagonistic fitness effects in males and females to drive chromosome degeneration is therefore limited. While some regions of the PAR had divergent male-female allele frequencies, suggestive of sexually antagonistic alleles, coalescent simulations showed this was broadly consistent with neutral genetic processes. Our results indicate that the degeneration of the large and ancient sex chromosomes of the ostrich may have been slowed by high recombination in the female PAR, reducing the scope for the accumulation of sexually antagonistic variation to generate selection for recombination cessation.

Experimental evidence that group size generates divergent benefits of cooperative breeding for male and female ostriches.

Cooperative breeding allows the costs of parental care to be shared, but as groups become larger, such benefits often decline as competition increases and group cohesion breaks down. The counteracting forces of cooperation and competition are predicted to select for an optimal group size, but variation in groups is ubiquitous across cooperative breeding animals. Here, we experimentally test if group sizes vary because of sex differences in the costs and benefits of cooperative breeding in captive ostriches, Struthio camelus, and compare this to the distribution of group sizes in the wild. We established 96 groups with different numbers of males (1 or 3) and females (1, 3, 4, or 6) and manipulated opportunities for cooperation over incubation. There was a clear optimal group size for males (one male with four or more females) that was explained by high costs of competition and negligible benefits of cooperation. Conversely, female reproductive success was maximised across a range of group sizes due to the benefits of cooperation with male and female group members. Reproductive success in intermediate sized groups was low for both males and females due to sexual conflict over the timing of mating and incubation. Our experiments show that sex differences in cooperation and competition can explain group size variation in cooperative breeders.

Being a parent is hard work. The unrelenting demand for food and protection is exhausting. Now imagine being a parent on the hot African savannah. Food and water are scarce, and whenever you leave your offspring, they overheat, or something eats them. This is the reality for ostriches. They, like humans, cope with the challenges of parenthood by sharing childcare responsibilities. Ostriches live in groups, breed in a communal nest, and take it in turns to incubate their eggs. This helps to maximize the survival of their offspring, but it has its downsides. The bigger a group gets, the more its members have to compete over mates and space for their eggs in the nest. The balance between cooperation and competition should, in theory, result in one 'optimal' group size. But this pattern does not seem to hold true: in the wild, ostrich families vary wildly in size and composition. To find out why, Melgar et al. set up dozens of groups of breeding ostriches and gave them different opportunities to cooperate. For males, there was one group size that maximized the number of offspring they produced (reproductive success): a single male with four or more females. Males did not benefit much from cooperation, and suffered greatly from competing with other males for mates. For females, however, the story was different. They benefited much more than males from cooperation and did best in bigger groups where they could share egg care with other individuals. Middle-sized groups were not good for either sex because reproduction was hard to coordinate: males continued to pursue copulations after females had initiated incubation, resulting in eggs being exposed and broken. The different priorities of males and females explain why there is no single optimal group size for ostriches. How groups balance competition and cooperation is a fundamental question in biology. Why do some organisms prefer to live alone, while others thrive in large groups? Understanding more about the balance of priorities within a group could hold the answers. It could also help to inform conservation work and animal breeding by showing how different social pressures influence breeding success.

Mass Sperm Motility Is Correlated to Sperm Motility as Measured by Computer-Aided Sperm Analysis (CASA) Technology in Farmed Ostriches.

Semen analyses have gained momentum in various livestock industries. However, in farmed ostriches, semen analysis is still in its experimental stage, and males are not screened for sperm quality before breeding. This study investigated the correlations between computer-aided sperm analysis (CASA) technology and the traditional, yet affordable, mass sperm motility score. Semen was collected from nine South African Black ostrich males (mean age ± SD: 5.25 ± 1.21 years), using the dummy female method for 5 consecutive days monthly, for 8 months. Mass sperm motility scores were recorded on a scale of 1−5 (1: little to no sperm movement; 5: rapid sperm movement). The CASA traits recorded were: total motility (MOT), progressive motility (PMOT), curve−linear velocity (VCL), straight-line velocity (VSL), average path velocity (VAP), amplitude of lateral head displacement (ALH), linearity (LIN), straightness (STR), wobble (WOB), and beat-cross frequency (BCF). The results revealed positive correlations between mass sperm motility and PMOT, MOT, VCL, and VAP ranging from 0.34 to 0.59 (p < 0.0001). In contrast, negative correlations were recorded between mass sperm motility and LIN, STR, and BCF, with correlations ranging from −0.20 to −0.39 (p < 0.0001). VSL, ALH, and WOB were not correlated to mass sperm motility (p > 0.05). Ostrich farmers may thus be able to evaluate sperm motility reliably and potentially select breeding males by using the affordable mass sperm motility scoring method. Determining the correlation between these methods and fertility after artificial insemination or natural mating is however needed.

Minimum sperm dose for optimal fertility after artificial insemination in ostriches.

Artificial insemination (AI) in ostriches may present potential solutions to high proportions of infertile eggs commonly recorded on commercial farms and assist in reducing the number of males for breeding purposes thereby leading to a more economical and efficient farming system. Although non-invasive methods to collect semen and to artificially inseminate female ostriches have been developed, the insemination dose for maximum fertility of eggs remains unknown. This study was thus conducted to determine the minimum sperm dose that would promote fertility of eggs following AI in female ostriches. A total of 22 South African black ostriches (7 males and 15 females) aged between 2 and 9 years old were used. Semen samples were collected using the dummy female method and diluted 1:4 (semen: diluent) with an ostrich specific semen diluent. Females were inseminated with various sperm doses of diluted semen from the same male three times a week, every second day resulting in a total sperm dose of A: <2.5 × 109, B: 2.5-5 × 109, C: 5-7.5 × 109 and D: 7.5-9.6 × 109 sperm/week. Eggs produced after insemination were opened to determine the fertilization status of the germinal disc (GD) with an unaided eye. The fertile period was then calculated as the number of days fertilized eggs were laid after the last AI. Furthermore, the number of sperm trapped in the outer perivitelline membrane (spermOPVL) above the GD region was counted under fluorescent light, following staining with 4',6-diamidino-2-phenylindole to determine the rate of sperm loss and the number of days up to when the last egg containing sperm was laid. On average, a mean (±sd) of 35.34 ± 25.72% eggs produced after AI were fertilized. Fertility was lower (mean ± se) when sperm dose A was used (6.71 ± 9.40%), as compared to sperm dose B (46.01 ± 6.71%), C (37.34 ± 6.60%) and D (37.75 ± 8.36%) (P < 0.05). No significant difference was recorded in the latter three doses (P > 0.05). Furthermore, the length of the fertile period and the rate of sperm loss did not differ significantly between sperm doses (P > 0.05). Hence, a sperm dose of between 2.5 and 5 × 109 sperm/week is recommended to optimize fertility after AI in ostriches, as increasing the sperm dose would not benefit fertility. Further studies are, however, needed to determine the frequency of insemination that would maintain fertility throughout the breeding season as well as hatchability of eggs laid after AI.

The effect of tree shade on ambient conditions and heat stress indicator traits of new-born South African Mutton Merino and Dormer lambs: Preliminary results.

This preliminary study investigated the provision of shade on heat stress indicators of South African Mutton Merino (SAMM) and Dormer lambs shortly after birth, during the autumn 2017 and 2018 lambing seasons. Newborn lambs were assessed to determine whether welfare, as assessed by respiratory response and rectal temperature as heat stress indicators, survival and early growth benefitted from the provision of shade. Groups consisting of 4-17 pregnant SAMM and Dormer ewes were randomly allocated to 5-10 paddocks with natural shade from trees and 5-9 paddocks that were directly in the sun with no shade available. The lambs were recorded within 24 h of birth at noon. Climate data were obtained from a nearby weather station. The lambs were also weighed at 12 (SD = 2) days of age at tail-docking. Tree shade had a moderating effect on temperature, resulting in lower maximum daytime and higher minimum night-time temperatures. There was an interaction between a temperature-humidity index (THI) and the treatments (access to shade or no access to shade) for respiration and rectal temperature (P < 0.01). Both traits were relatively unaffected by the THI at values below 77. Unshaded lambs exhibited a pronounced upwards trend following a THI-threshold of 77-78. Tailing weight tended to be higher while lamb survival of live-born lambs to tail-docking was lower in lambs born in shaded paddocks but these trends did not persist to weaning. Shade is needed to enhance animal welfare by alleviating the effect of high THI-values on hot days in an autumn lambing season.

Long term genetic selection for reproductive success affects neonatal lamb vitality across cold stress conditions.

Adverse weather conditions are important contributors to mortality in new-born lambs. Previous studies have shown variation between lambs in their ability to cope with circumstances of cold stress, and genetic selection could be a viable option for improving animal robustness. The Elsenburg Merino flock was divergently selected on number of lambs weaned (NLW). This resulted in divergent responses in reproduction and lamb survival. This study evaluated lamb vitality and mortality of positively selected H-Line relative to the negatively selected L-Line in response to cold stress. Traits included lamb rectal temperature (RT), surface temperature (ST), shiver score (SS), lamb vigor score (LVS), breaths per minute (BPM), mortality to three days of age (M3) and to weaning (TM). Cold stress was described by a chill index derived from daily rainfall, wind speed and ambient temperature, and represented as the mean of the one (CI), two (CI-2) or three (CI-3) days since parturition. Overall, H-Line lambs had a higher neonatal RT and were less likely to succumb than L-Line contemporaries. In a significant (P < 0.05) interaction, the predicted RT of L-Line showed a non-linear decline with increased levels of CI-2, while H-Line lambs better maintained their core temperature. M3 was also affected by a significant interaction between CI-3 and selection line, further suggesting that observed lower mortality rates in the H-Line depends on H-Line lambs' improved ability to cope with stressful environments. Long term selection for NLW in the H-Line led to improvements in both adaptations associated with lower lamb losses. The continued recording of viability traits to produce larger datasets amenable to genetic analysis is recommended, specifically for rectal temperature.

Expression of cytokine genes at tick attachment and control sites of Namaqua Afrikaner, Dorper and South African Mutton Merino sheep.

Cytokines are immune response components important in innate immunity and inflammatory response. They are harnessed as part of local immunological responses by animals to combat local infections and/or infestations. This study investigated expression of four selected cytokine genes, namely, interleukin 1 beta (IL-1ß), chemokine C-C ligand 2 (CCL2), chemokine C-C ligand 26 (CCL26) and interleukin 8 (IL-8), at tick attachment and control sites in a South African indigenous sheep breed the Namaqua Afrikaner (NA) and two commercial breeds, the Dorper and South African Mutton Merino (SAMM). The NA was previously shown to be more resistant to infestation by ticks than the two commercial breeds. NA ewes expressed IL-1ß more at tick attachment sites compared to Dorpers. The NA breed was also more likely to upregulate the expression of the CCL2, CCL26 and IL-8 genes at tick attachment sites compared to control sites than the other breeds. The results of this study gave an indication that cytokines are involved in immune responses to tick challenge and laid a foundation for further studies under controlled challenge conditions.

Extreme temperatures compromise male and female fertility in a large desert bird.

Temperature has a crucial influence on the places where species can survive and reproduce. Past research has primarily focused on survival, making it unclear if temperature fluctuations constrain reproductive success, and if so whether populations harbour the potential to respond to climatic shifts. Here, using two decades of data from a large experimental breeding programme of the iconic ostrich (Struthio camelus) in South Africa, we show that the number of eggs females laid and the number of sperm males produced were highly sensitive to natural temperature extremes (ranging from -5 °C to 45 °C). This resulted in reductions in reproductive success of up to 44% with 5 °C deviations from their thermal optimum. In contrast, gamete quality was largely unaffected by temperature. Extreme temperatures also did not expose trade-offs between gametic traits. Instead, some females appeared to invest more in reproducing at high temperatures, which may facilitate responses to climate change. These results show that the robustness of fertility to temperature fluctuations, and not just temperature increases, is a critical aspect of species persistence in regions predicted to undergo the greatest change in climate volatility.

The effect of dilution rate and successive semen collections on sperm quality and sexual motivation of sexually mature South African Merino rams.

This study investigated whether dilution rate and successive semen collections influenced sperm viability, morphology, motility and male sexual motivation in sexually mature South African Merino rams (SAMR). Semen was collected from 11 rams up to either sperm or behavioural exhaustion. Semen was then immediately serial diluted to make 0, 2, 4, 8 and 16× dilutions with Hams F10 diluent. Following dilution, sperm motility was evaluated using computer-assisted sperm analysis (SCA®), while sperm morphology and viability were assessed using nigrosin-eosin staining and SYBR14/PI, respectively. Male sexual motivation was recorded by reaction time to first mount, while male dexterity was calculated as the ratio of mounts to ejaculations. An increase of dilution rate did not affect sperm motility (P > 0.05) but resulted in a significant decrease in the percentage of live normal sperm (P < 0.05). Furthermore, while sperm concentration and number decreased with semen collection number (P < 0.05), no effect was detected on sperm viability, morphology and motility (P > 0.05), except for average curvilinear velocity which showed a biphasic trend (P < 0.05). Mating success and reaction time were negatively affected by successive semen collections (P < 0.05), while male dexterity was unaffected (P > 0.05). Nevertheless, relatively high numbers of motile sperm (>2 billion) were collected up to the 4th successive semen collection, with a short initial reaction period (<25 s) and good success rate (>65%). SAMR thus withstood frequent semen collections without affecting their sperm reserves or sexual motivation. Further studies are required to investigate optimal conditions for semen collection and artificial insemination in this breed.

Extensive human presence and regular gentle handling improve growth, survival and immune competence in ostrich chicks.

A total of 416 day-old ostrich chicks were randomly allocated to one of the three different husbandry practices for 3 months after hatch; HP1 (extensive human presence with gentle human voice, visual and gentle physical stimuli), HP2 (similar to HP1 but without physical stimuli) and S (human presence limited to supply of feed and water). Chick weight (kg) was measured at 6 and 12 weeks of age, while mortalities were recorded daily to calculate the survival rate. Finally, chicks' antibody responses to vaccination against Newcastle disease (NCD) was measured using the Hemagglutination-Inhibition (HI) test at 20 weeks of age. While HP1 chicks were heavier and survived better to 6 weeks of age than HP2 and S chicks (p < .05), no difference was observed thereafter (p > .05). Furthermore, HP1 chicks had an improved immune competence, as illustrated by their lower percentage of positive HI titers, compared to HP2 and S chicks (p < .05). Hence, integrating extensive human presence with positive human-chick interactions may assist in alleviating challenges related to chick rearing in the ostrich industry.

The Effect of Extensive Human Presence at an Early Age on Stress Responses and Reactivity of Juvenile Ostriches towards Humans.

The effect of extensive human presence and regular gentle handling performed at an early age (0⁻3 months old) on stress responses and reactivity of juvenile ostriches towards humans was investigated. A total of 416 ostrich chicks over two years were exposed to one of three treatments for three months after hatching; namely, Human Presence 1 (HP1, N = 144): extensive/prolonged human presence with physical contact (touch, stroking), gentle human voice, and visual stimuli; Human Presence 2 (HP2, N = 136): extensive/prolonged human presence without physical contact, but with gentle human voice and visual stimuli; and the Standard treatment (S, N = 136): human presence limited to routine feed and water supply as a control. At 7.5 months of age, the plasma heterophil/lymphocyte (H/L) ratio was measured before and 72 h after feather harvesting and feather clipping to determine acute stress responses, while chronic stress was measured by quantification of corticosterone (CORT) concentrations in the floss feathers of the birds. Birds' behavioural response towards a familiar or an unfamiliar handler was evaluated at 12 months using docility and fear tests, and through behavioural observations conducted on random days between the ages of 8⁻13 months. Willingness to approach, and to allow touch interactions, aggressiveness, and exhibition of sexual display towards the handler, was recorded. No difference in the H/L ratios before and after feather harvesting and clipping was observed in HP1 birds, whereas H/L ratios showed a significant increase 72 h post feather harvesting and clipping in HP2 and S birds (p < 0.05). Birds from the S treatment exhibited a significantly (p < 0.05) higher feather CORT concentration compared with HP1 birds, while HP2 birds had intermediate responses. Birds' reactivity towards humans and temperament as evaluated using behavioural observations, docility, and fear tests was not affected by treatment (p > 0.05). However, HP1 and HP2 birds were more inclined (p < 0.05) to approach a familiar rather than an unfamiliar handler during the behavioural observations, indicating an ability to distinguish between a familiar and an unfamiliar handler. Overall, the results indicate that early gentle human interactions with ostrich chicks can be beneficial in reducing physiological stress sensitivity later in life and facilitate the ability of ostriches to distinguish between familiar and unfamiliar handlers.

Genetic diversity and population structure of South African smallholder farmer sheep breeds determined using the OvineSNP50 beadchip.

A population structure study was performed in South African ovine populations using the OvineSNP50 beadchip. Blood samples were obtained from 295 sheep of which 172 had been identified as smallholder Dorpers, 4 smallholder White Dorpers, 46 purebred Dorpers, 26 purebred South African Mutton Merinos and 47 purebred Namaqua Afrikaners. Blood from the latter three breeds were obtained from a resource flock maintained on the Nortier research farm. Genetic diversity was estimated using allelic richness (A r), observed heterozygosity (H o), expected heterozygosity (H e) and inbreeding coefficient (F). Population structure analysis was performed using fastSTRUCTURE to determine the breed composition of each genotyped individual. The Namaqua Afrikaner had the lowest H e of 0.280 ± 0.18 while the H e of smallholder Dorper, Dorper and South African Mutton Merino did not differ and were 0.364 ± 0.13, 0.332 ± 0.16 and 0.329 ± 0.17, respectively. The average inbreeding coefficient was highest for the pure breeds, Namaqua Afrikaner, Dorper and South African Mutton Merino compared to the average inbreeding coefficient for the smallholder Dorper population. The smallholder Dorper were introgressed with Namaqua Afrikaner, South African Mutton Merino and White Dorpers. Similarly, the smallholder Dorper population was more genetically diverse than the purebred Dorper, South African Mutton Merino and Namaqua Afrikaner from the research farm. The higher genetic diversity among the smallholder sheep may be advantageous for their fitness and can be used to facilitate selective breeding.

Genetic variation in and relationships among faecal worm eggs recorded in different seasons of the year at the Tygerhoek farm in South Africa.

Gastrointestinal nematodes result in severe economic and production losses to the sheep industry. An increase in resistance of the nematodes to chemicals used for control, as well as a demand of consumers for meat products free from chemicals, has fostered research on alternative control strategies. Breeding for resistance to nematodes offers an alternative to control parasitism but its effectiveness depends on genetic variation in faecal worm egg count (FWEC), an indirect measure of parasite resistance. A historic dataset of FWEC from four Merino lines subjected to natural parasite challenge was used to estimate genetic parameters for FWEC in three seasons (autumn, winter and spring) using a repeated records animal model, followed by a three-trait animal model analysis treating FWEC in different seasons as separate traits. The effects of selection line, birth year, sex, the sex x birth year interaction, season and the season x year interaction were significant when using 4994 records recorded from 1997 to 2000 (p < 0.001). The heritability of log-transformed FWEC amounted to 0.09 ± 0.02, with no contribution from the animal permanent environmental variance to the between animal variation across seasons. Three-trait heritability estimates for log-transformed FWEC amounted to 0.07 ± 0.05 in autumn, 0.13 ± 0.05 in winter and 0.19 ± 0.05 in spring. These results suggest sufficient genetic variation in FWEC to support selection for lower log-transformed FWEC. However, the best time to record data for selection is after the break of the season in winter and in spring, when sheep are stimulated by a greater intake of infective larvae from the pasture after the first rains. Genetic correlations among FWEC in the respective seasons were moderate to high, ranging from 0.55 to 0.89. Phenotypic correlations, on the other hand, were significant but lower in magnitude, ranging from 0.09 to 0.16. These results provide useful information for developing strategies for the genetic improvement of ovine resistance to gastrointestinal nematodes under Mediterranean conditions in South Africa by using FWEC as an indicator trait.

Developing a female-only flock for artificial insemination purposes in ostriches: Progress and future directions.

The development of a flock of females that can produce eggs and maintain egg production rate without the presence of males is a prerogative for a viable artificial insemination protocol in ostriches. Over six consecutive breeding seasons (May-December, 2009-2014), we recorded the egg production performance of 40 single-penned (ART) South African Black ostrich females (2-9 years of age), and compared these records with the egg production of 162 pair-mated females of comparable age from the breeding flock (BP). ART females laid significantly fewer eggs than BP females (mean±SEM: 3.49±0.13 eggs per month vs. 4.64±0.09 eggs per month respectively; P<0.001). Both groups showed a similar pattern of laying, with a peak production in July to September. The mean egg weight of ART females was significantly lower than those of BP females (1367±2.25g vs. 1423±1.1g, respectively; P<0.001). Furthermore, female age significantly affected egg production and egg weight whereby BP females reached a peak egg production at 3 years of age, while in ART females, egg production was the highest at 5 years of age. Interestingly, the number of eggs produced, clutches and eggs per clutch of ART females were independent of visual stimulation from the males. These results indicate that male presence is not needed to ensure egg production. Continuous recruitment of young females based on human-friendly behaviour to breeding by artificial insemination from high egg production performance parents could improve egg production of the ART flock. Studies are also needed to gain a better understanding of underlying physiological mechanisms promoting spontaneous ovulation in this species.

Predicting ejaculate quality and libido in male ostriches: effect of season and age.

The success of artificial breeding program depends largely on the reproductive performance of males. Male performance can vary with season and age impacting on quality and quantity of semen collected for artificial insemination purposes and therefore fertility of inseminated females. We examined variation in semen output and male libido of seven male ostriches (aged 2-5 years) over a period of 24 months. We collected ejaculates using a dummy female and measured semen characteristics (ejaculate volume, sperm concentration, number of spermatozoa per ejaculate, sperm motility and morphology) and male libido (willingness to mount the dummy). A total of 1006 ejaculates were collected. Across months, the volume of semen (mean ± SEM) ranged from 1.03 ± 0.12 mL to 1.85 ± 0.07 mL, the sperm concentration from 3.21 ± 0.12 × 10(9)/mL to 4.16 ± 0.74 × 10(9)/mL, and the number of spermatozoa from 3.42 ± 0.28 × 10(9) to 7.66 ± 0.47 × 10(9). The largest volume of ejaculates and the highest number of sperm were collected in spring. Ejaculates with higher number of normal sperm were also collected in spring-early summer, whereas ejaculates with higher numbers of live abnormal and dead sperm were collected in winter. Sperm motility was relatively constant over months, despite a reduction in summer (January-February), while male libido peaked in winter (June-July) and spring (October-November). Furthermore, we observed high individual variation between males for all variables tested, except for motility. These results indicate that collections conducted in spring yield higher number of spermatozoa, when the libido of males is also at a maximum. Therefore in this species seasonal variation in semen quality should be considered in breeding programmes by artificial insemination to maximise fertility.

The effect of temperature and pH on the motility and viability of ostrich sperm.

As the chemical environment of semen diluents can have a profound effect on sperm quality, we examined the effect of temperature and pH on the motility and viability of sperm in the ostrich. Semen was collected from four males, each male being replicated three times. Ejaculates were diluted and incubated for 10 min at 20°C and 40°C in four different buffers, temperature adjusted at pH 6, 7, 8 and 9 respectively. Average path velocity (VAP), curvilinear velocity (VCL), straight-line velocity (VSL), linearity (LIN), beat cross frequency (BCF) and amplitude of lateral displacement (ALH) were then recorded for each sample using CASA. The viability of sperm was assessed using nigrosin-eosin staining. Sperm incubated at 40°C had higher motility parameters, except for ALH. At 40°C, VAP, VSL and LIN increased with pH while VCL, BCF and ALH were higher for lower pHs. The viability of sperm was not affected by temperature but decreased at pH values>7. A pH in the neutral range appeared to yield higher quality sperm after in vitro storage at 20°C. However, the effect of different pH levels and temperatures on sperm longevity needs to be investigated further to develop viable ostrich specific diluents.

Twice daily collection yields greater semen output and does not affect male libido in the ostrich.

The success of an artificial insemination program in ostriches is highly dependent on the yield of viable semen. We, therefore, tested how semen output is affected by three different collection frequencies: once every 2d (48h interval), daily (24h interval), and twice a day (6h interval). Ejaculates were collected from seven male ostriches (aged 2-4 years) for 10 consecutive days using the dummy female method. We assessed semen characteristics (sperm motility, volume, concentration, number of sperm per ejaculate and sperm viability) and male libido (the delay between the presentation of the dummy and ejaculation, and the willingness to mount the dummy). The total daily output of semen and the number of sperm were greater at the 6h collection interval than at the 24h or 48h interval while sperm motility and viability were not affected. At the 6h interval, the number of live normal sperm increased over the treatment period while the number of live abnormal sperm was reduced. Furthermore, the time that males took to mount the dummy and their willingness to copulate with the dummy were unaffected by collection frequency. Across males we observed great individual variation in both semen characteristics and libido suggesting there is the potential to increase the efficiency of semen collection by selecting superior males. These results indicate not only that two collections per day yield maximum semen output and may improve semen viability, but also that quantifying variation between males may help further increase semen collection efficiency.