Respiratory and cardiovascular responses to acute hypoxia and hyperoxia in internally pipped chicken embryos.

During the first day of hatching, the developing chicken embryo internally pips the air cell and relies on both the lungs and chorioallantoic membrane (CAM) for gas exchange. Our objective in this study was to examine respiratory and cardiovascular responses to acute changes in oxygen at the air cell or the rest of the egg during internal pipping. We measured lung (VO2(lung)) and CAM (VO2(CAM)) oxygen consumption independently before and after 60 min exposure to combinations of hypoxia, hyperoxia, and normoxia to the air cell and the remaining egg. Significant changes in VO2(total) were only observed with combined egg and air cell hypoxia (decreased VO2(total)) or egg hyperoxia and air cell hypoxia (increased VO2(total)). In response to the different O2 treatments, a change in VO2(lung) was compensated by an inverse change in VO2(CAM) of similar magnitude. To test for the underlying mechanism, we focused on ventilation and cardiovascular responses during hypoxic and hyperoxic air cell exposure. Ventilation frequency and minute ventilation (V(E)) were unaffected by changes in air cell O2, but tidal volume (V(T)) increased during hypoxia. Both V(T) and V(E) decreased significantly in response to decreased P(CO2). The right-to-left shunt of blood away from the lungs increased significantly during hypoxic air cell exposure and decreased significantly during hyperoxic exposure. These results demonstrate the internally pipped embryo's ability to control the site of gas exchange by means of altering blood flow between the lungs and CAM.     

Effects of Shiny Cowbird Molothrus bonariensis parasitism on different components of House Wren Troglodytes aedon reproductive success

Avian brood parasites, including cuckoos and cowbirds, have multiple negative effects on their hosts. We analysed the effects of Shiny Cowbird Molothrus bonariensis parasitism on different components (e.g. egg losses, hatching success, chick survival and nest abandonment) of House Wren Troglodytes aedon reproductive success. We also conducted an experiment to discriminate between two mechanisms that may reduce hatching success in parasitized clutches: lower efficiency of incubation due to the increase in clutch volume and disruption of host incubation by the early hatching of Cowbirds. Egg puncturing by Shiny Cowbirds reduced host clutch size at hatching by 10–20%, and parasitized nests had a decrease in hatching success of 40–80%. Egg losses and hatching failures were positively associated with the intensity of parasitism. Brood reduction was greater in parasitized nests, but the growth rate of the chicks that fledged was similar to that in unparasitized nests. The combined effects of egg losses, hatching failures and brood reduction decreased the number of fledged chicks by 80%. In addition, egg puncturing increased the likelihood of nest abandonment by Wrens. Experimental data showed that hatching failures occurred when there was a combination of: (1) an increase in the volume of the clutch by the addition of the Cowbird egg without removal of host eggs, and (2) the addition of the Cowbird egg before the onset of incubation. This was relatively common in House Wren nests, as Cowbirds generally parasitize before the onset of incubation. Our results indicate that Shiny Cowbird parasitism imposes a major impact on House Wrens, as it affects all components of the Wren's reproductive success.     

Experimental analysis on the variations of acid-base balance in the chick embryo

1. The pH remains steady, 7.57. 2. The pCO2 increases gradually. We believe this is fundamental for hatching. 3. Bicarbonate and base excess increase from acidosis to alkalosis due to absorption of the egg shell. 4. The pO2 increases in stage 41 and 42 due to pulmonary respiration. 5. The pO2 drop prior to hatching due to involution of the chorioalantoid membrane and increasing requirements.     

Effects of posture on the respiratory mechanics of the chick embryo

In the chicken embryo, pulmonary ventilation and pulmonary gas exchange begin approximately one day before the completion of hatching. We asked to what extent the posture inside the egg, and the presence of the eggshell and membranes, may alter the mechanical behaviour of the respiratory system. The passive mechanical properties of the respiratory system were studied in chicken embryos during the internal pipping phase (rupture of the air cell) or the external pipping phase (hole in the eggshell). Tracheal pressure and changes in lung volume were recorded during mechanical ventilation, first, with the embryo curled up inside the egg, then again after exteriorization from the eggshell. In the internal pippers, respiratory system compliance increased and expiratory resistance decreased after exteriorization, whereas the mean inspiratory impedance did not change. In the external pippers, exteriorization had no significant effects on respiratory compliance, resistance, or impedance, and the values were similar to those of newly hatched chicks. We conclude that, in the chicken embryo, at a time when pulmonary ventilation becomes an important mechanism for gas exchange, the curled up posture inside the egg does not provide any significant mechanical constraint to breathing.     

Fertility and hatchability of eggs laid in the pullet-to-breeder transition period and in the initial production period

The initial eggs produced by broiler breeder hens are relatively small compared with later in the production cycle. An evaluation of indices related to hatchability is required when these eggs are to be used for the production of broiler chicks. Two experiments were conducted to evaluate characteristics related to the hatchability of eggs from pullet-to-breeder transition phase, at 25 and 27 weeks of age, and from the peak of production period and five weeks later, at 32 and 37 weeks of age. Eggs from birds 25 weeks had a lesser fertility in Experiment 1. Mortality occurred unevenly in early (1–5 days), middle (6–17 days) and late (18–21 days) incubation, and greater mortality was observed after the internal membrane was ruptured. The younger the hen, the lighter the egg, chick, and shell, and the longer the time required to complete the hatching process. In Experiment 2, greater mortalities were observed at the early period (1–5 days) and after “pipping” of the internal and external membranes. Embryos from heavy eggs of breeder hens 37 weeks of age took less time to complete the hatching process. Results indicated the larger the egg, the heavier the chick and shell, and the lesser the shell percentage. As breeder age advanced, characteristics related to egg fertility and hatchability improved.     

Moisture during Egg Incubation Adversely Affects Hatching of the West Indian Sweetpotato Weevil,Euscepes postfasciatus (Coleoptera: Curculionidae), When the Eggs are Incubated En Masse

We compared the egg-hatching of Euscepes postfasciatus (Fairmaire) with and without moisture treatment to test whether moisture is necessary during egg incubation when a large number of eggs are gathered into a mass. Moisture treatment exhibited significant undesirable effects on hatching (reduction of hatchability, delay of hatching, and increase of the variance of hatching date) compared to no moisture treatment. In addition, moisture treatment significantly increased the incidence of fungus on the egg surface, which can subsequently contaminate the larval artificial diet. Based on these results, we concluded that moisture is not necessary for incubating E. postfasciatus eggs. Two possible explanations for the undesirable effects of moisture on hatching were discussed: a direct effect by preventing respiration of the eggs and an indirect effect through fungal infection of the eggs.     

Egg temperature and initial brood patch area determine hatching asynchrony in Magellanic penguin Spheniscus magellanicus

In birds, the adaptive significance of hatching asynchrony has been under debate for many years and the parental effects on hatching asynchrony have been largely assumed but not often tested. Some authors suggest that hatching asynchrony depends on the incubation onset and many factors have been shown to influence hatching asynchrony in different species. Our objective was to analyze the exact timing of the onset of incubation and if this affects hatching asynchrony; and, in addition, which other factors (brood patch development, incubation position, adult body condition, intra‐clutch egg dimorphism, laying date and year) affect hatching asynchrony in Magellanic penguins Spheniscus magellanicus. We first estimated the eggshell temperature at which embryo development starts, with a non‐destructive and novel method. We then recorded individual egg temperatures in 61 nests during incubation, and related them, and other breeding parameters, to hatching asynchrony. We also observed incubation positions in 307 nests. We found a significant positive relationship between hatching asynchrony and the temperature that the first‐laid egg experienced during egg laying and between hatching asynchrony and the initial brood patch area. We also found a negative relationship between hatching asynchrony and the difference in temperature between second and first‐laid eggs within a clutch, measured after the egg‐laying period was finished. We ruled out position of the eggs during incubation, adult body condition, egg volume, laying date, and study year as factors influencing hatching asynchrony. The egg temperature during laying and the difference in temperature between eggs of a clutch are determinants of hatching asynchrony in Magellanic penguins.     

A Meta-Analysis of Experiments Linking Incubation Conditions with Subsequent Leg Weakness in Broiler Chickens

A series of incubation and broiler growth studies were conducted using one strain of broiler chicken (fast feathering dam line) observing incubation effects on femoral bone ash % at hatch and the ability of the bird to remain standing at 6 weeks of age (Latency-To-Lie). Egg shell temperatures during incubation were consistently recorded. Parsimonious models were developed across eight studies using stepwise multiple linear regression of egg shell temperatures over 3-day periods and both bone ash at hatch and Latency-To-Lie. A model for bone ash at hatch explained 70% of the variation in this factor and revealed an association with lower egg shell temperatures during days 4–6 and 13–15 and higher egg shell temperatures during days 16–18 of incubation. Bone ash at hatch and subsequent Latency-To-Lie were positively correlated (r = 0.57, P<0.05). A model described 66% of the variation Latency-To-Lie showing significant association of the interaction of femoral ash at hatch and lower average egg shell temperatures over the first 15 days of incubation. Lower egg shell temperature in the early to mid incubation process (days 1–15) and higher egg shell temperatures at a later stage (days 16–18) will both tend to delay the hatch time of incubating eggs. Incubation profiles that resulted in later hatching chicks produced birds which could remain standing for a longer time at 6 weeks of age. This supports a contention that the effects of incubation observed in many studies may in fact relate more to earlier hatching and longer sojourn of the hatched chick in the final stage incubator. The implication of these outcomes are that the optimum egg shell temperature during incubation for broiler leg strength development may be lower than that regarded as ideal (37.8°C) for maximum hatchability and chick growth.     

Environmental and developmental effects on external gill loss in the red-eyed tree frog, Agalychnis callidryas

I examined the effects of development, hatching, and oxygen availability on external gill loss in red-eyed tree frogs, Agalychnis callidryas. Under natural conditions, the arboreal embryos maintained large external gills until hatching, which occurred from 5-8 d after oviposition. At hatching, when tadpoles entered the water, external gills began to regress. In older hatchlings this process was extremely rapid. Gill circulation was lost on average within 16 min and sometimes within 5 min. Gills often regressed completely in under 2 h. Younger hatchlings reduced gill circulation, shortened and adducted their gills, then resumed normal circulation for some time after hatching; half had completely lost external gills within 24 h. Experimentally increasing the area of egg surface exposed to the air induced loss of external gills in unhatched embryos. Older hatchlings in hypoxic water without access to air maintained their external gills. This suggests that loss of external gills is a response to increased oxygen availability, rather than a response to hatching per se. Extended maintenance of external gills by large, late-hatching embryos may facilitate continued rapid development in closely packed eggs.     

AN ACCOUNT OF THE HATCHING STRATEGIES OF BIRDS

1. Three basic hatching methods are described together with one subsidiary method. The symmetrical method is characterized by rotation of the chick in the egg during hatching climax; a line of damage around the circumference of the egg is evident at the beginning of a pushing phase which causes a fairly symmetrical cap to be broken from the shell. The asymmetrical method is used by a few long-billed species; it involves little or no rotation of the chick in the egg, and produces asymmetrical shell remains. The megapodes have developed a unique hatching method in response to their unusual incubation conditions. Parental assistance has been observed in some species, but only as an auxiliary to either the symmetrical or asymmetrical method. Approximately 150 species have been categorized according to the hatching method(s) they use. 2. Among those species adopting the symmetrical method, there is considerable variability as to how far the chick turns in the egg during hatching climax. On this basis, a spectrum of behaviour, expressed in terms of angle of rotation (θ), has been proposed. At one end lie species such as the bobwhite quail and little owl (θ≥ 360°), and at the other, the ostrich and rhea (θ≤ 90°). 3. The theory that, with the exception of the megapodes, there is only one basic hatching method is examined. The tenets of this theory are found to be inconsistent with recent observations of species differences in hatching behaviour. It is concluded that hatching behaviour is governed by an intrinsic species-specific programme, in turn influenced by mechanical or other external factors. 4. The literature contains several suggestions as to the external factors that might influence hatching technique, but only one detailed investigation. It is proposed that interspecific differences in the mechanical properties of the egg integument (the shell and its underlying membranes) can be regarded as forming a spectrum from very brittle to comparatively tough. The amount of climax rotation by the chick is seen as an adaptive response to brittleness or toughness of the egg integument. The hatching technique of the chick is further modified as a response to the effect of moisture content on the integument. 5. The selective pressures leading to the evolution of the symmetrical and asymmetrical hatching methods are discussed. The previous model for the development of the asymmetrical method is amended to account for those species of gull which may adopt either method.     

Individual birds advance offspring hatching in response to increased temperature after the start of laying

In seasonally reproducing organisms, timing reproduction to match food availability is key to individual fitness. Ambient temperature functions as an important cue for the timing of the food peak in temperate-zone birds. After laying start, individual birds may still improve synchrony between offspring hatching and food availability by adjusting the onset of incubation to most up-to-date cues about the development of the food source. However, it is unknown whether individuals respond to changes in temperature after the onset of laying, and whether individuals adjust incubation onset independent of clutch size. Here, I show in free-living blue tits (Cyanistes caeruleus) that experimental heating of nestboxes in the laying phase resulted in increased duration of nocturnal incubation bouts prior to clutch completion, leading to earlier hatching of eggs and increased hatching asynchrony. Experimental heating did not affect the number of laying gaps, egg volume and clutch size, nor were any carry-over effects on offspring detected. These results are best explained as a response to increased temperature acting as a cue for an advanced food-peak, rather than a relief of energetic constraints, because improved energetic conditions would not favour more hatching asynchrony. Other benefits cannot be excluded, since increased laying-phase incubation under warmer conditions may also help maintain egg viability. This study is the first to show that temperature has a causal effect on the time between clutch completion and hatching of the first offspring, indicating that behavioural adjustment to climate change can continue after laying start.     

Developmental consequences of association with a photosynthetic substrate for encapsulated embryos of an intertidal gastropod

Aggregation of embryos in clutches that lack internal circulation can increase the risk of hypoxia by limiting gas exchange. As a result, limits on oxygen solubility and diffusion in water can constrain the size and embryo concentration of aquatic egg clutches. Hypoxia in egg masses can slow embryo development, increase mortality, and reduce size at hatching. The risk of hypoxia for embryos, however, can be reduced by association with photosynthetic organisms. We examined whether embryo development in egg ribbons of the cephalaspidean mollusk Haminoea vesicula is significantly influenced by oviposition on eelgrass (Zostera marina). Association with the photosynthetic substrate had marked effects on development relative to association with non-photosynthetic substrates, and the direction of these effects was mediated by light conditions. Under intermediate and high light levels, association with eelgrass accelerated embryo development, while under dim light, the presence of the macrophyte increased development rate and reduced hatchling shell size. Benefits of association with eelgrass at higher light levels likely result from oxygen production by eelgrass photosynthesis, while we attribute costs under low light to oxygen depletion by eelgrass respiration. Association with Z. marina also limited microphyte growth in egg ribbons of H. vesicula. In the field, measurements of light attenuation within an eelgrass bed showed that conditions under which benefits accrue to embryos are ecologically relevant and correspond to spatial patterns of oviposition on eelgrass in the field. The choice of a photosynthetic oviposition substrate under appropriate light conditions can improve embryo fitness by accelerating embryo development without compromising hatchling size and by reducing the potential for excessive and harmful fouling by microphytes.     

Comparative analysis of the development of the lizard,Liolaemus tenuis tenuis. II. A series of normal postlaying stages in embryonic development

In this work, we have completed a study of the development of the ovoviviparous lizard Liolaemus tenuis tenuis. Ovoviviparity in this lizard is a condition in which eggs are retained within the reproductive duct for about 60 days. During this period the phases of segmentation, gastrulation, neurulation, presomitic, and somitic embryos transpire. During the months of December and January the eggs are laid, and at this time the embryos are comparable to stage 27 Liolaemus gravenhorsti lizard embryos, or to stage 29 Calotes versicolor lizard embryos. Differentiation of the facial region occurs between Days 12 and 42 after egg laying. Limbs develop rapidly between the 8th and 23rd days. By 53 days the appendicular skeleton is completely formed. After 36 days the mesonephros begins to degenerate, and its function is gradually taken over by the developing metanephros. Newborn lizards do not possess an egg caruncle. During the period up to hatching, there is a great increase of liquid within the egg, presumably amniotic fluid. Cracks develop in the leathery shell shortly before hatching and are, perhaps, the first sign of the onset of hatching. Increase of liquid in the egg during postlaying development accounts for its increase in weight and change in shape. Weight of the embryo at hatching does not exceed 32% of the total weight of the egg.     

Characterisation of proteases involved in egg hatching of the sheep blowfly, Lucilia cuprina

A number of proteases were identified in the egg shell washings (ESW) collected during the egg hatching of Lucilia cuprina (sheep blowfly). Characterization of these proteases indicated a pH optima in a similar pH range that was optimal for L. cuprina egg hatching. Mechanistic characterization of these proteases indicated that they were predominantly of the serine class. Several protease inhibitors were tested for their ability to inhibit L. cuprina egg hatching in vitro. Egg hatching was significantly (P<0.05) inhibited by PMSF (61%), 1,10-Phenanthroline (42%) and Pepstatin (29%). The inhibition of egg hatching by PMSF showed a strong concentration dependence, with its effects ranging from inhibition at high concentrations to enhancement of egg hatching at low concentrations. Addition of ESW to unhatched eggs, significantly (P<0.05) enhanced their rate of hatching above untreated control eggs. This enhancement of egg hatching was significantly (P<0.05) reversed by the protease inhibitors Elastatinal (40%), 1,10-Phenanthroline (40%) and PMSF (38%). These studies indicate a role for serine and/or metallo-proteases in facilitating L. cuprina egg hatch.     

Effects of temperature, salinity, and air exposure on development of the estuarine pulmonate gastropod Amphibola crenata

The primitive pulmonate snail Amphibola crenata embeds embryos within a smooth mud collar on exposed estuarine mudflats in New Zealand. Development through hatching of free-swimming veliger larvae was monitored at 15 salinity and temperature combinations covering the range of 2-30 ppt salinity and 15-25 °C. The effect of exposure to air on developmental rate was also assessed. There were approximately 18,000 embryos in each egg collar. The total number of veligers released from standard-sized egg collar fragments varied with both temperature and salinity: embryonic survival was generally higher at 15 and 20 °C than at 25 °C; moreover, survival was generally highest at intermediate salinities, and greatly reduced at 2 ppt salinity regardless of temperature. Even at 2 ppt salinity, however, about one-third of embryos were able to develop successfully to hatching. Embryonic tolerance to low salinity was apparently a property of the embryos themselves, or of the surrounding egg capsules; there was no indication that the egg collars protected embryos from exposure to environmental stress. Mean hatching times ranged between 7 and 22 days, with reduced developmental rates both at lower temperature and lower salinity. At each salinity tested, developmental rate to hatching was similar at 20 and 25 °C. At 15 °C, time to hatching was approximately double that recorded at the two higher exposure temperatures. Exposing the egg collars to air for 6-9 h each day at 20 °C (20 ppt salinity) accelerated hatching by about 24 h, suggesting that developmental rate in this species is limited by the rates at which oxygen or wastes can diffuse into and from intact collars, respectively. Similarly, veligers from egg capsules that were artificially separated from egg collars at 20 °C developed faster than those within intact egg collars. The remarkable ability of embryos of A. crenata to hatch over such a wide range of temperatures and salinities, and to tolerate a considerable degree of exposure to air, explains the successful colonization of this species far up into New Zealand estuaries.     

Timing matters: species‐specific interactions between spawning time,substrate quality,and recruitment success in three salmonid species

Substratum quality and oxygen supply to the interstitial zone are crucial for the reproductive success of salmonid fishes. At present, degradation of spawning grounds due to fine sediment deposition and colmation are recognized as main factors for reproductive failure. In addition, changes in water temperatures due to climate change, damming, and cooling water inlets are predicted to reduce hatching success. We tested the hypothesis that the biological effects of habitat degradation depend strongly on the species‐specific spawning seasons and life‐history strategies (e.g., fall‐ vs. spring‐spawners, migratory vs. resident species) and assessed temperature as an important species‐specific factor for hatching success within river substratum. We studied the species‐specific differences in their responses to such disturbances using egg‐to‐fry survival of Danube Salmon (Hucho hucho), resident brown trout (Salmo trutta fario), and migratory brown trout (Salmo trutta lacustris) as biological endpoint. The egg incubation and hatching success of the salmonids and their dependence on temperature and stream substratum quality were compared. Hatching rates of Danube salmon were lower than of brown trout, probably due to higher oxygen demands and increased interstitial respiration in spring. Increases in maximum water temperature reduced hatching rates of resident and migratory brown trout (both fall‐spawners) but were positively correlated with hatching rates of Danube salmon (a spring‐spawner). Significantly longer incubation periods of resident and migratory brown trout coincided with relatively low stream substratum quality at the end of the egg incubation. Danube salmon seem to avoid low oxygen concentrations in the hyporheic zone by faster egg development favored by higher water temperatures. Consequently, the prediction of effects of temperature changes and altered stream substratum properties on gravel‐spawning fishes and biological communities should consider the observed species‐specific variances in life‐history strategies to increase conservation success.     

A novel high throughput assay for anthelmintic drug screening and resistance diagnosis by real-time monitoring of parasite motility

Background

Helminth parasites cause untold morbidity and mortality to billions of people and livestock. Anthelmintic drugs are available but resistance is a problem in livestock parasites, and is a looming threat for human helminths. Testing the efficacy of available anthelmintic drugs and development of new drugs is hindered by the lack of objective high-throughput screening methods. Currently, drug effect is assessed by observing motility or development of parasites using laborious, subjective, low-throughput methods.

Methodology/Principal Findings

Here we describe a novel application for a real-time cell monitoring device (xCELLigence) that can simply and objectively assess anthelmintic effects by measuring parasite motility in real time in a fully automated high-throughput fashion. We quantitatively assessed motility and determined real time IC₅₀ values of different anthelmintic drugs against several developmental stages of major helminth pathogens of humans and livestock, including larval Haemonchus contortus and Strongyloides ratti, and adult hookworms and blood flukes. The assay enabled quantification of the onset of egg hatching in real time, and the impact of drugs on hatch rate, as well as discriminating between the effects of drugs on motility of drug-susceptible and –resistant isolates of H. contortus.

Conclusions/Significance

Our findings indicate that this technique will be suitable for discovery and development of new anthelmintic drugs as well as for detection of phenotypic resistance to existing drugs for the majority of helminths and other pathogens where motility is a measure of pathogen viability. The method is also amenable to use for other purposes where motility is assessed, such as gene silencing or antibody-mediated killing.     

Trans-shell infection by pathogenic micro-organisms reduces the shelf life of non-incubated bird's eggs: a constraint on the onset of incubation?

Many birds initiate incubation before clutch completion, which results in asynchronous hatching. The ensuing within-brood size disparity often places later-hatched nestlings at a developmental disadvantage, but the functional significance of the timing of the onset of incubation is poorly understood. Early incubation may serve to maintain the viability of early-laid eggs, which declines over time owing to the putative effects of ambient temperature. An unexplored risk to egg viability is trans-shell infection by micro-organisms. We experimentally investigated the rate and magnitude of microbial trans-shell infection of the egg, and the relative effects of ambient temperature and micro-organisms on hatching success. We show that infection of egg contents is prevalent and occurs within the time required to lay a clutch. The probability of infection depends on the climatic conditions, the exposure period and the phylogenetic composition of the eggshell microbiota. We also demonstrate that microbial infection and ambient temperature act independently to reduce egg viability considerably. Our results suggest that these two factors could affect the onset of avian incubation in a wide range of environments.     

Egg size variation in birds with asynchronous hatching: is bigger really better?

In many animals large size at birth enhances offspring survival, but comparative evidence remains equivocal for birds. Failure to consider asynchronous hatching (ASH) may have confounded previous analyses. We assessed effects of egg size and ASH on growth and survival of common grackle (Quiscalus quiscula) nestlings to test the hypothesis that females adjust the size of last-laid eggs to modify effects of ASH. Although positive, the effect of egg size on nestling growth and survival was overwhelmed by the effect of ASH, with late-hatched nestlings being most likely to starve. Egg size did significantly affect growth late in the nestling period, but only because starvation had greatly reduced hatching asynchrony among surviving nestlings. Similarly, in experimentally synchronized nests, egg size and hatching asynchrony both affected offspring growth early in the nestling phase. Our results suggest that there is unlikely to be an adaptive advantage to females from varying the size of last-laid eggs in species with substantial ASH and that studies to assess the effect of a given maternal effect (e.g., varying egg size) should be done in the context of other maternal effects that may be operating simultaneously (e.g., ASH).     

Cryopreservation of the milt of the northern pike

Seven published extenders, three thawing media and two thawing temperatures were tested in order to determine their suitability for cryopreservation of northern pike ( Esox lucius L.) sperm. Sperm motility during successive steps of cryopreservation was evaluated. Erdahl and Graham's extender with the addition of egg yolk proved to be the most efficient (maximum hatching rate of 74.5%) when semen was thawed in 120 m m NaCl solution warmed up to 30° C. No correlations between motility of sperm (diluted in extenders or diluted in extenders and then activated with thawing solution) and the subsequent hatching success were observed. The relationship between motility of thawed sperm and its fertilization ability was considerable, but correlation was not significant. Spermatozoa frozen in some extenders were frequently motile after thawing but they were not able to fertilize the eggs, this resulted in a poor hatching rate. Depending on the extender, the addition of yolk induced either positive or detrimental effects on fertilization success.