Artificial Incubation Of Eggs Research Articles

Influence of temperature on embryonic development of Pontastacus leptodactylus freshwater crayfish, and characterization of growth and osmoregulation related genes

Narrow clawed crayfish, Pontastacus (Astacus) leptodactylus, represents an ecologically and economically valuable freshwater species. Despite the high importance of artificial breeding for conservation purpose and aquaculture potential, hatching protocols have not been developed so far in this species. Further, limited knowledge exists regarding the artificial egg incubation, the temperature effect on embryonic development, hatching synchronization and hatching rate. In the present study we investigated the temperature increase (from 17 oC to 22oC) effects in two different embryonic developmental stages of P. leptodactylus. Furthermore, two primer pairs for the Fibroblast Growth Factor Receptor 4 (FGFR4) gene cDNA amplification were successfully designed, characterising for the first time the FGFR4 gene in P. leptodactylus in relation to different developmental stages and temperatures. Apart from the FGFR4 gene, the Na+/K+-ATPase α-subunit expression was also explored. Both the FGFR4 and Na+/K+-ATPase α-subunit expression levels were higher in embryos closer to hatching. Egg incubation at 22oC for seven days led to significant increase of FGFR4 expression in embryos from earlier developmental stages. Nevertheless, temperature increase did not affect FGFR4 expression in eggs from latter developmental stages and Na+/K+-ATPase α-subunit expression in all developmental stages. Temperature increase represents therefore probably a promising strategy for accelerating hatching in freshwater crayfish particularly in early developmental stages. Specifically, our results indicate that FGFR4 expression increased in embryonic stages closer to hatching and that temperature influences significantly its expression in embryos from earlier developmental stages. Overall, these findings can provide a better understanding of artificial egg incubation of P. leptodactylus, and therefore can be employed for the effective management of this species, both for economic and biodiversity retention reasons.

Artificial incubation of eggs of the Eastern Montpellier Snake (Malpolon insignitus) and physical abnormalities in hatchlings

Artificial incubation of eggs of the Eastern Montpellier Snake (Malpolon insignitus) and physical abnormalities in hatchlings

Breeding history and husbandry of the Raggiana Bird-of-paradise (Paradisaea raggiana).

The Raggiana Bird-of-paradise (Paradisaea raggiana) has been held by the San Diego Zoo for more than 50 years, but the population remained in low numbers until the mid-1990s. A previous article published in 1997 documented the beginning of the rise in the population; however, no new information has been published since that time. Over the past 25 years, behavioral observations by animal staff have led to improvements in husbandry techniques, which includes artificial incubation of eggs and hand-rearing of chicks. The ability to simulate a lek with various housing arrangements, in which the males can display with each other, be given access to a female for copulation, and then be separated again has proven paramount for breeding success. Additionally, females are allowed mate choice, further mimicking natural behaviors observed by this species. The refinement of these methods has led to a greater number of fertile eggs as well as higher hatchability and survivability, which in turn has resulted in a significant increase in the captive population.

Effects of Sibling Eggs Contact on Incubation Length and Hatching Synchrony in Red-Legged Partridge (Alectoris rufa)

Hatching synchrony is a desirable trait in the artificial incubation of eggs because it permits the simultaneous extraction of all the chicks from the hatcher, thus minimizing extraction queues. This study aimed at evaluating the effects on egg performance, incubation length, and hatching synchrony of different sibling contact regimes during the incubation in red-legged partridge (Alectoris rufa) eggs. To achieve these objectives, 196 eggs were arranged in four treatments according to the sibling eggs contact regime: (a) eggs without contact during the whole incubation process; (b) eggs in contact during the incubation phase (from day 1 to 20 of incubation); (c) eggs in contact during the hatching phase (from day 20 of incubation to hatching); (d) eggs in permanent contact throughout the incubation process (from day 1 of incubation to hatching). Development stage at embryonic mortality, hatchability, and egg weight loss during incubation were not affected, but incubation length was shortened and hatching synchrony was increased in eggs in contact during the hatching phase. The main conclusion was that keeping red-legged partridge eggs in close contact during the hatching stage in the artificial incubation does not affect hatchability but allows hatching synchrony to be maximized, facilitating the handling of 1 day old chicks.

A male-specific sex marker for the endangered western sawshelled turtle (Myuchelys bellii) using in silico whole-genome subtraction

Artificial incubation of eggs for the mass release of hatchlings is a common conservation intervention for imperilled turtle species. Programs designed to reinforce wild populations need to ensure that they are releasing appropriate male to female ratios into the wild. In many turtle species, the sex of juveniles cannot be identified using external morphology until they approach sexual maturity. For the endangered western sawshelled turtle, Myuchelys bellii, sexual dimorphism does not occur until at least 6 years of age. We aimed to develop a molecular test to identify the sex of M. bellii during the life stages where they cannot be sexed morphologically—embryos, hatchlings and small juveniles. We used in silico whole-genome subtraction of a female M. bellii (XX) from a male (XY) to identify a Y chromosome-specific sequence which we characterized and developed into a PCR sex test. Our research is the first to use a whole-genome subtraction method in-silico to successfully establish sex chromosome markers in a freshwater turtle species. Developing this technology provides an opportunity for conservation programs to ensure that populations are supplemented with a proportionate number of male and female hatchlings. Further, it allows large scale measurement of naturally occurring sex ratios in hatchlings and small juveniles, which in turn enables estimates of sex ratios within wild populations free from age-at-maturity bias. The application of sex-specific marker technology also provides an opportunity to quantify the influence of sex on behaviour, movement and survival in the segment of populations that cannot be morphologically sexed.

Optimising the hatching success of artificially incubated eggs for use in a conservation program for the western saw-shelled turtle (

Artificial incubation of eggs and the release of hatchlings into the wild is a common conservation intervention designed to augment threatened turtle populations. We investigate a range of incubation temperatures to establish an optimal temperature for maximum hatching success of western saw-shelled turtle (Myuchelys bellii) eggs. We report on the influence of incubation temperature on incubation duration and hatching success and describe two experimental incubation methods which, for the same incubation temperature (27°C), resulted in 77% and 97% hatching success, respectively. Eggs were incubated at constant temperatures (27°C, 28°C and 29°C) to determine the influence of temperature on incubation period, hatchling morphology and external residual yolk. Incubation duration was negatively correlated with incubation temperature. We report on the morphology of eggs and hatchlings and show that their dimensions are positively correlated with maternal adult size and mass. A constant incubation temperature of 27°C produced the highest hatching success and smallest external residual yolk on hatching and is therefore recommended for incubation of eggs for population reinforcement programs. Our study is the first to optimise artificial incubation procedures for M. bellii and will be a valuable resource for M. bellii and other threatened freshwater turtle conservation initiatives.

A Study of the Impact and Challenges Faced using Artificial Egg Incubation

A Study of the Impact and Challenges Faced using Artificial Egg Incubation

A CONTROL SYSTEM FOR REMOTE MONITORING AND CONTROL OF THE TEMPERATURE IN CHICKEN EGGS HATCHING USING ARTIFICIAL INCUBATORS

According to OACD/FAO Agricultural Outlook, poultry meat remains the primary driver of growth in total meat production. Its short production cycle allows producers to respond quickly to market needs, as well as rapid improvements in genetics, animal health, feeding practices, breeding conditions and breeding environment. Hatching process needs to be done properly in order to maximize the number of healthy chickens hatched and to minimize their death rate. When using artificial egg incubation, it is important to monitor and control ambient conditions in incubators, to obtain ventilation and egg turning. Ambient conditions are expressed in terms of temperature and humidity, where maintaining and controlling the temperature inside the incubator is of the greatest importance. This paper deals with the description of a control system developed for remote monitoring and control of the temperature in chicken eggs hatching using artificial incubators. The system consists of heater in a form of an incandescent light bulb, temperature sensor, relay, microcontroller, and communication module. For temperature control, a PID (proportional-integral-derivative) controller is designed, which maintains the temperature at 37.8 °C. Temperature readings are available through the web browser and are accessible using wired Internet connection. Proposed system represents a simple, low-cost but very efficient solution which provides the necessary conditions for proper development of chickens while growing inside the egg.

How deep is deep enough? Analysis of sea turtle eggs nest relocation procedure at Chagar Hutang Turtle Sanctuary.

Sea turtle eggs incubation involves natural and artificial incubation of eggs, and indeed the depth will be varied and presumably affect the development of hatchlings. For nest relocation, the researcher needs to decide on the depth to incubate the eggs. Sea turtle eggs clutches may vary between 40 and 120 eggs for the green turtle, thus using a single value as the standard procedure might affect the quality of hatchlings. Here we quantify the dimension of the natural (in-situ) nest constructed by the nester and the artificial (ex-situ) built by our ranger during nest relocation. We suggest a linear regression calculation of Y = 0.2366X + 59.3267, better predict a more accurate nest depth based on the number of eggs to imitate the natural nest.

Moving from trends to benchmarks by using regression tree analysis to find inbreeding thresholds in a critically endangered bird.

Understanding how inbreeding affects endangered species in conservation breeding programs is essential for their recovery. The Hawaiian Crow ('Alalā) (Corvus hawaiiensis) is one of the world's most endangered birds. It went extinct in the wild in 2002, and, until recent release efforts starting in 2016, nearly all of the population remained under human care for conservation breeding. Using pedigree inbreeding coefficients (F), we evaluated the effects of inbreeding on Hawaiian Crow offspring survival and reproductive success. We used regression tree analysis to identify the level of inbreeding (i.e., inbreeding threshold) that explains a substantial decrease in 'Alalā offspring survival to recruitment. Similar to a previous study of inbreeding in 'Alalā, we found that inbreeding had a negative impact on offspring survival but that parental (vs. artificial) egg incubation improved offspring survival to recruitment. Furthermore, we found that inbreeding did not substantially affect offspring reproductive success, based on the assumption that offspring that survive to adulthood breed with distantly related mates. Our novel application of regression tree analysis showed that offspring with inbreeding levels exceeding F = 0.098 were 69% less likely to survive to recruitment than more outbred offspring, providing a specific threshold value for ongoing population management. Our results emphasize the importance of assessing inbreeding depression across all life history stages, confirm the importance of prioritizing parental over artificial egg incubation in avian conservation breeding programs, and demonstrate the utility of regression tree analysis as a tool for identifying inbreeding thresholds, if present, in any pedigree-managed population.

ЭФФЕКТ ВОЗДЕЙСТВИЯ ПЕРЕМЕННОЙ ТЕМПЕРАТУРЫ ИНКУБАЦИИ НА РОСТ И МЕТАБОЛИЗМ КУРИНЫХ ЭМБРИОНОВ ЯИЧНЫХ КРОССОВ И РАЗВИТИЕ ИХ ВИСЦЕРАЛЬНЫХ ОРГАНОВ

The article discusses issues related to the impact of variable incubation temperature (37.8°C – from the 1st to the 14th day; 39.5°C, two hours daily, in the 15th-17th days; 37.5°C – 18th day; 37.0°C – 19th-21st day; relative humidity is 57.0%) on the growth and metabolic rates of the Hysex Brown and Lohmann Brown crosses chicken embryos as well as on the development of their visceral organs and the incubation effectiveness itself. Currently, most of the research is devoted to studying the influence of variable egg incubation temperatures while using one cross of chickens and comparison with the standard incubation mode. A necessary condition for the balanced development of productive bird embryos is the acquisition of knowledge about their morphophysiological parameters and metabolic needs, based on which it is possible to adjust regimes of artificial egg incubation. The research results have shown that the developed and tested incubation regime with variable temperatures is most effective for hen eggs of the Lohmann Brown cross compared to the Hysex Brown cross. This positive effect was expressed in a more intensive growth of chicken embryos and the development of some visceral organs (gizzard stomach, spleen), as well as in the most pronounced rate of gas exchange (CO2), the highest energy value of embryos and the lowest total energy consumption for growth and metabolism in the period from 4 days of incubation to the hatching time. Differentiation of the temperature regime during embryogenesis has a positive effect on incubation results. The percentage of withdrawal of the Lohmann Brown cross young chickens is 4% higher than that of the Hysex Brown cross due to decreased embryonic mortality and decreased number of frozen embryos and suffocated chickens. This kind of research, in our opinion, is an auspicious one, and it is necessary to adjust the existing incubation regimes of hen eggs in various production areas.

Determination of Artificial Incubation Time of Some Malawi Cichlid Species Incubating in the Mouth (Iodotropheus sprengerae, Cyrtocara moorii, Maylandia estherae, Labidochromis caeruleus) Eggs

Malawi cichlid species belonging to the Cichlidae family are among the most popular commercial species in the aquarium industry. Females of this species begin to incubate their eggs in the mouth after ovulation. Professional producers continue to induce vomiting of the eggs from the female's mouth at many different times and grow them with artificial incubation. The aim of this study is to determine the most appropriate time to induce vomiting and artificial incubation of eggs of these species. For this purpose, rusty cichlid (Iodotropheus sprengerae), blue dolphin cichlid (Cyrtocara moorii), red zebra cichlid (Maylandia estherae) and electric yellow cichlid (Labidochromis caeruleus) were produced in colonies. The development of eggs and larvae obtained from broodstocks were observed. Critical times for cichlid culture have been determined. While electric yellow cichlid (L. caeruleus) completed its embryonic development on the 3rd day after ovulation, blue dolphin cichlid (C. moorii) and red zebra cichlid (M. estherae) species completed on the 4th day, rusty cichlid (I. sprengerae) completed on the 5th day. Therefore, the results of the present study revealed that it is not appropriate to apply the same incubation technique to all these species.

Insights into first feeding of newly-hatched paralarvae of the merobenthic octopus, Octopus tetricus

The commercial aquaculture of merobenthic octopus species is constrained by the inability to raise large numbers of paralarvae through their planktonic phase from hatching to juvenile. Knowledge on the early feeding behaviour and nutrition requirements of octopus paralarvae is critical for addressing this challenge. This study evaluates the food seizure behaviour of Octopus tetricus paralarvae at age 0–5 dph (days post-hatch) under different experimental conditions including, egg incubation method, tank colour, and live prey versus formulated food pellets. The percentage of paralarvae that attacked food items and the percentage of paralarvae that had at least one successful seizure at the age 0–5 dph were measured in relation to the experimental conditions. The number of attacks and successful seizures of food items by paralarvae was not affected by the egg incubation method, i.e., maternal grooming versus artificial incubator tank. A higher percentage of paralarvae performed attacks in black tanks (70 ± 8%) compared to grey tanks (44 ± 6%) with no differences to white tanks (58 ± 7%) when Artemia nauplii were supplied. However, no differences in the proportion of paralarvae with successful seizures were found among the three tested tank colours. A higher percentage of paralarvae attacked formulated food pellets (69 ± 6%) compared to Artemia nauplii (44 ± 6%) in grey tanks. An overall trend was observed of O. tetricus paralarvae having lower successful seizures at 0 dph that increased until 2 to 3 dph followed by a steep and continuously decline in success through until 5 dph. On average 31 ± 3% of paralarvae had at least one successful seizure of formulated food pellets in the first 5 min after the pellets were supplied compared to 4 ± 1% in the subsequent 10 min. In contrast, feeding on Artemia nauplii remained steady over the full 15 min experimental period. The results indicate that the effect of tank colour on feeding behaviour warrants further investigation as it affects the ability of the paralarvae to detect and attack potential food items. Moreover, the results demonstrate that artificial egg incubation did not have a negative effect on the feeding behaviour of paralarvae, and that formulated food pellets have the potential to be developed as an alternative to live prey for the culture of paralarvae of this species.

The exploration of artificial incubation of Cherax quadricarinatus eggs

The redclaw crayfish, Cherax quadricarinatus, is an economically valuable freshwater crayfish. However, some production obstacles, such as a low egg hatching rate and asynchronous hatching, are hindering its development in the aquaculture industry. Artificial incubation of eggs may solve these problems. This study explored the technology of artificial incubation of redclaw crayfish eggs. The following results were obtained: 1) 75% alcohol as a disinfectant for 60 s had a preferable antibacterial effect and promoted the hatching rate; 2) densities of 300 and 600 eggs/incubator resulted in significantly higher hatching and survival rates than a density of 900 eggs/incubator; 3) at a density of 600 eggs/incubator, the optimum number of net pieces for attachment of freshly hatched juveniles was 20 per incubator; 4) with a density of 600 eggs/incubator and 20 net pieces/incubator, the hatching rate was 82.05% ± 4.09%, the survival rate was 55.12% ± 7.51%, and a total of 129,200 SPF (specific pathogen-free) seedlings were cultivated. This artificial incubation system was maintained at close to an aseptic state, with an absence of white spot virus, iridovirus, Vibrio and ciliates; this was true for all water sources used and for incubation of both eggs and juvenile crayfish. In conclusion, if we implement reasonable methods of disinfection, SPF detection and pathogen isolation and utilize optimal egg densities and incubation systems, large-scale production of SPF seedlings of Cherax quadricarinatus is possible.

Using the heart rate of a bird embryo to monitor its condition

The article deals with the problem of artificial incubation of eggs of bird species, whose incubation is carried out for the first time and for which there are no data on the optimal temperature regime. The question of the need for using the methods allowing individual monitoring of the embryo is raised. The data from the literature on non-invasive methods of biological control of incubation, allowing changing the heart rate of a bird embryo to control its condition, are presented. It was found that the heart rate of embryos decreases as they develop. A similar effect on the heart rate, regardless of age, provides a decrease in temperature and lack of oxygen. Motor activity of the embryo creates interference check of the heart rate. The results of testing the device for determining the heart rate of the embryo of birds by the optical method are presented. The circle of factors influencing the accuracy of measurements is defined. The possibility of lifetime determination of the duration of hypoxia of the embryo of birds by monitoring the heart rate is experimentally confirmed. The possibility of using the method of heart rate control to detect the degree of hypoxia of the embryo in order to determine the exact time of human operational intervention in the process of hatching of rare bird species is discussed.

Assessing conservation tools for an at-risk shorebird: Feasibility of headstarting for American OystercatchersHaematopus palliatus

SummaryManagement of threatened and endangered populations of wildlife increasingly relies upon active intervention such as predator control, habitat manipulation, andex situbreeding or care. One tool that has received consideration for the management of declining or threatened avian populations is headstarting, or the artificial incubation of eggs and subsequent placement of newly hatched chicks in original or foster nests. We assessed the feasibility of implementing a headstarting program for the American OystercatcherHaematopus palliatus, a species of high conservation concern in the eastern USA. Annual productivity is often low and lost during incubation, suggesting artificial incubation could enhance annual productivity. We used a control-impact approach to assign nests as either control or headstart and measured daily survival rate, success of parents accepting headstarted chicks, attendance patterns and behaviours of parents, and chick survival. We also implemented a transparent scoring process to rate the success of each step and the overall program. Daily survival rates of nests were significantly higher at headstart compared to control nests, and parents continued to incubate when eggs were well secured at nest sites. Attendance patterns and behaviour did not differ between headstart and control parents, and parents readily accepted healthy chicks whether they were returned to original or foster nests. Chick survival and subsequently annual productivity were, however, not higher at headstart compared to control nests suggesting that although we were able to enhance nest survival, low chick survival was still limiting annual productivity. Ultimately, headstarting may be most appropriate for American Oystercatchers where productivity is lost primarily to flooding, predation, or disturbance during the incubation stage but not during the chick-rearing stage. If, for example, high rates of nest loss are due to predators that also may prey upon chicks, then headstarting may not be an effective conservation tool.

Результаты реализации антиоксидантных свойств композиции растворов коламина и витамина C в период эмбриогенеза у мясных кур

В статье дана комплексная оценка эмбрионов и цыплят контрольных и опытных групп, полученных из яиц, обработанных композицией растворов коламина и витамина C, по ряду важнейших интерьерных и биохимических показателей развития в условиях промышленной инкубации. Необходимость повышения вывода кондиционных цыплят возникла одновременно с появлением искусственной инкубации яиц, что в значительной степени отличается от естественного насиживания, поэтому даже при использовании самого современного оборудования в условиях искусственной инкубации эмбрион испытывает значительные стрессы, вызывающие чрезмерное образование свободных радикалов. Для их нейтрализации всё чаще используют естественные метаболиты. Наибольший позитивный эффект достигнут при трансовариальном использовании. Эксперименты проводили в условиях птицефабрики «Михайловский бройлер» на яйцах кур кросса «Хаббард-F15». Все яйца были подобраны по принципу аналогов с учетом времени снесения, сроков хранения и массы. В каждую партию входило по 300 яиц. Экспериментальную партию яиц перед закладкой в инкубатор и при переводе на вывод обрабатывали ранее выявленной оптимальной концентрацией композицией растворов коламина и витамина С, при этом проводили комплекс исследований по общепринятым методикам. Установлена тенденция к стимуляции фосфолипидного обмена, что выразилось в достоверном увеличении фосфатидилхолина на 26,3 % (p < 0,01) и в повышении уровня общих липидов на 2,9 %. Таким образом, можно предположить, что коламин как составляющая композиции предложенных нами препаратов проникает в яйцо — к эмбриону и в дальнейшем используется организмом для построения каркаса клеточных мембран.

Captive Husbandry and Socialization of the Red‐Billed Chough (Pyrrhocorax pyrrhocorax)

Since the 1970s, Paradise Park Wildlife Sanctuary in Cornwall, United Kingdom, has built up a captive flock of red-billed choughs Pyrrhocorax pyrrhocorax and over 30 years has developed successful methods of keeping, breeding, and appropriately socializing them in captivity. A total of 77 nests reached the egg stage with 27 nests producing at least one young and 48 young fledging in total. Several components are important in achieving successful breeding and socialization. Provision of live food, especially ant's eggs and small mealworms and crickets, in the first days after hatching is essential, improving the condition of adults and survival of nestlings. Situating aviaries in quiet areas, away from public view, is important. Socialization in family groups during the winter months and allowing choughs to choose partners induces better compatibility. Introduction of nest cameras greatly improved young survival through early identification of health problems enabling treatment of young between hatching and 10-days old, when mortality is otherwise highest, and enabling precautionary medication shortly after hatching. We show that clutch size increases significantly with female age and that direct intervention such as artificial egg incubation and hand rearing can be successful and worthwhile, but its requirement is reduced by closer monitoring. Red-billed choughs provide a good model species to further develop captive management and release techniques that can then be applied to critically endangered species that show similar social and long-learning behaviors. Captive breeding programs can play an important role in such work through provision of suitable birds and supporting avicultural expertise.

Artificial incubation, egg replacement and adoptive parents in bird management: a test with Lesser Elaenia Elaenia chiriquensis

SummaryReducing the time that birds’ eggs are exposed during incubation in the wild is a management strategy with the potential to reduce nest predation rates, enhance breeding success and increase the population size of endangered species. We tested whether manipulation of clutches through artificial incubation of Lesser Elaenia Elaenia chiriquensis eggs and subsequent reintroduction of new-born nestlings to their original nest, and the use of adoptive parents, were efficient ways of increasing the population size of this species. We evaluated the financial cost and benefit of three different management strategies of artificial egg incubation with reintroduction of nestlings to the original nest. We searched for nests and manipulated eggs during the breeding seasons of 2009 and 2010 in a savanna reserve in central Brazil. Real eggs were replaced by artificial eggs and artificially incubated. The following breeding parameters were monitored: hatching rate, fledgling productivity, daily survival rate of nestlings and nest success. The effect of nest monitoring frequency (daily or every 3–4 days) on breeding parameters was also tested. Hatching rate was much higher amongst artificially incubated eggs than naturally incubated eggs. Artificially incubated clutches presented higher rates of fledgling production and apparent nest success than non-manipulated clutches. Clutch manipulation did not interfere with nestling daily survival rate. Daily monitoring did not have negative effects. The clutch manipulation methodology we used proved to be viable and is a potential tool for increasing population size.

Sodium chloride as effective antifungal treatment for artificial egg incubation inAustropotamobius pallipes

In this study, sodium chloride at three different concentrations, 30 000 ppm (S30), 60 000 ppm (S60) and 90 000 ppm (S90), and formaldehyde at one concentration, 3000 ppm (F), were tested as antifungal chemicals during artificial incubation (AI) of Austropotamobius pallipes eggs. Two treatments were tested without chemicals as control groups with (R) and without (C) the removal of dead eggs. After AI, formaldehyde treatment ensured high survival of stage 1 (89.7 ± 2.3%) and stage 2 (85.5 ± 2.4%) of juveniles. However, comparable survival rate to stage 1 and stage 2 (85.5 ± 5.5% and 80.6 ± 3.2%) were also found in the treatment with the highest sodium chloride concentration (S90). Significantly lower survival rate of juveniles (stage 1: 60.6–70.3% and stage 2: 56.1–67.3%) were evident in groups S60, S30 and R. However, group R demanded high labor and related costs. The lowest juvenile survival levels to stage 1 (46.4 ± 8.2%) and stage 2 (45.2 ± 6.8%) were observed in treatments without fungicide chemicals and removal of dead eggs (C).