Influence of various pre-starter diets on growth and the development of the small intestine of ostrich chicks 

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Chapter 2 The composition of egg yolk absorbed by starved ostrich (Struthio camelus L.) chicks from one to seven days post-hatching and for ostrich (Struthio camelus L.) chicks from one to sixteen days post-hatching on a pre-starter broiler diet

Introduction

In the early 20^th century, studies were conducted to determine the effect of feeding on yolk assimilation in chickens (Roberts, 1928; Heywang & Jull, 1930). The chicks were starved for about 48 to 72 hours post-hatching to allow the yolk to be completely absorbed and prevent possible bowel problems which were thought to occur when yolk assimilation took place while exogenous food was fed simultaneously (Heywang & Jull, 1930). Since then, various studies investigated the effect of feeding and starvation on yolk utilisation and the development of the digestive tract and enzyme activity in poultry (Bierer & Eleazer, 1965; Chamblee et al., 1992; Noy et al., 1996; Noy & Sklan, 1998; Dipner, 2000; Asya et al., 2003; Zehava, 2003). It was concluded that yolk utilisation was more efficient in chicks that received feed and water after hatching and that starvation has a negative effect on the development of the digestive tract and digestive enzymes. Much less work has been done on yolk utilisation in ostrich chicks (Bertram & Burger, 1981; Deeming, 1995; Mushi et al., 2004), especially with regard to the influence of feeding and starvation on the effect of yolk utilisation.
In this chapter, two separate trials were conducted. The first trial was to determine the chemical composition of the yolk of starved ostrich chicks, one to seven days post-hatching. The second trial was to determine the chemical composition of the yolk of ostrich chicks fed a broiler pre-starter diet which could conceivably affect the uptake of the yolk and stimulate growth and development of the digestive tract and digestive enzymes. The two trials are not compared, as there were too many variables in the methodology of the trials. The project had ethical approval from the Onderstepoort Animal Use and Care Committee (Protocol 36-5-623).

Materials and Methods

Animals

The first trial involved 35 ostrich chicks. Fertilized eggs were obtained from the Oudtshoorn Experimental farm of the Department of Agriculture Western Cape, South Africa and transported to the Faculty of Veterinary Science of the University of Pretoria, Onderstepoort, South Africa. Eggs were moved on day 36 of incubation. The eggs were packed by a professional ostrich egg transport company in suitable containers which eliminated excessive shaking and cooling down of the eggs during transport, but allowed sufficient ventilation. Should the egg temperature fall below the critical temperature of 25°C, or excessive shaking occur, increased shell deaths would result. The hatchability of eggs decreases by 5-10% during the transportation process and the chicks in such cases normally need assistance with hatching, as they tend to get weaker, due to lower oxygen and higher carbon dioxide levels in the containers (Danie Terblanche, 2009, personal communication).
Eggs were hatched in a poultry incubator at 36°C with the humidity set at 24%. Eggs that did not hatch within the first two days of arrival at Onderstepoort were manually cracked to assist with hatching. Those chicks were slaughtered in the trail first, as they tended to be weaker than the others (Zanell Brand, 2003, personal communication). To make up for losses sustained, eight day-old chicks were obtained from a nearby ostrich farm to maintain a sufficient number of birds for the trial. Chicks were kept in a clean, disinfected room which was kept cool and dark. Noise and human contact were restricted to the minimum to limit stress. Chicks were provided with clean drinking water, but no food. Five chicks were slaughtered each day for seven consecutive days, starting from the day of hatching (day one). The mean hatching and slaughter weights of the starved chicks, as well as the yolk weight and pH, are presented in Table 1.
During the second trial, six chicks were slaughtered every second day (except on day 8 when 7 chicks were slaughtered) over a period of 16 days. The first group of chicks were thus two days old when sacrificed and a total of 49 chicks were slaughtered. The chicks were obtained from the same source as for the first trial and similarly handled and transported to Onderstepoort. The average weight of the day-old chicks was 839.3 g.
The trial continued for 16 days as it was found that yolk was still present in 14-day old chicks (Adriaan Olivier, 2005, personal communication). Chicks were kept under similar conditions as in the first trial, except for the provision of a poultry pre-starter diet (AFGRI Animal Feeds, South Africa). The minimum specifications for the commercial pre-starter diet were: protein 24.5%, moisture 11.5%, energy 12 MJ/kg feed, fat 6.7%, crude fibre 3.6% and ash 6.2%. The values for the fed chicks are presented in Table 2.

Measurements and Calculations

In both trials, chicks were euthanized with CO₂ in a closed container. The yolks sacs were immediately removed from the carcasses, weighed and pH values determined (Thermo Orion pH meter, Orion Research, Inc.500 Cummings Centre, Beverly, MA 01915, USA). As much yolk as possible was taken from the yolk sac for proximate analysis (ash, dry matter, crude protein and ether extract analyses) and immediately frozen at -20°C.
Yolk samples from both trials were freeze-dried and analyzed for ash, dry matter (DM), crude protein (CP) content and fat content (measured as ether extract (EE)) by standard methods (AOAC, 1995).
Yolk samples from the first trial (the starved chicks) were further analyzed for amino acids, fatty acids and glucose composition. As the same tendencies were observed for crude protein and fat values in both trials, the analyses for amino acids, fatty acids and glucose composition were not repeated for the second trial due to insufficient funds and were assumed to be similar.
Amino acids were determined on freeze-dried samples by ion-exchange chromatography of the acid-hydrolysed protein. Samples were hydrolysed (AOAC, 1995) with 6M HCl in a sealed tube under N₂ for 22 hours in an oil bath at 110°C and then stored at -20°C. On the day the analyses were done, the samples were thawed to room temperature. Each sample was then mixed by vortex for 5-10 seconds and centrifuged at 15 000 g for 5 minutes in a Hermle bench centrifuge (HERMLE Labortechnik GmbH, Wehingen). The supernatant (25 µl) was placed in a glass hydrolysis tube and dried under vacuum for 1 hour.
The pH was adjusted to pH=7 by adding 20 µl methanol : water : triethylamine 2:2:1 and the samples were re-dried for 1 hour. Each sample was derivatised by adding 20 µl derivatising solution (methanol : water : triethylamine : phenylisothiocyanate (PITC) 7:1:1:1). The mixture was incubated at room temperature for 10 min. and then dried under vacuum for a minimum of 1 hour and a maximum of 3 hours until completely dry. The derivatised dried sample was dissolved in 400 µl of Picotag® sample diluent (Waters, Millford, MA, USA), filtered through a 0.45 uM filter and 16 µl of sample was subjected to HPLC using a standard method for PTC-amino acid chromatography.
Data was collected and analysed using Breeze software (Waters, USA). The percentage recovery of standards was determined by analysing standards of a known quantity. For each batch of samples at least two standards were dried and treated under the exact conditions as the samples. Fatty acid methyl esters (FAME) were prepared according to the method of Morrison & Smith (1964). The FAME were analysed with a GLC: Varian Model 3300, equipped with flame ionisation detection and two 30m fused silica mega bore DB-225 columns of 0.53 mm internal diameter (J & W Scientific Folsom, CA). Gas flow rates were: hydrogen, 25 ml/min; air, 250 ml/min. and nitrogen (carrier gas), 5-8 ml/min. Temperature programming was linear at 4°C/min; initial temperature, 160°C; final temperature, 220°C held for 10 min.; injector temperature, 240°C and detector temperature, 250°C. The FAME were identified by comparison of the retention times to those of a standard FAME mixture (Nu-Chek-Prep Inc., Elysian, Minnesota) and the milligram fatty acid per gram tissue sample was calculated.
Glucose content was determined using the ACE™ Glucose Reagent (Reagent number NAE2-27) intended for the quantitative determination of glucose in serum using the ACE™ clinical chemistry system. In the ACE Glucose method, glucose is determined after enzymatic oxidation in the presence of glucose oxidase. The resultant hydrogen peroxide reacts under catalysis of peroxidase with phenol and 4-aminophenazone to form a red-violet quinoneime dye as an indicator. The absorbance of the reaction is bi-chromatically measured at 505 nm/692 nm.

Statistics

The experimental design for the first trial entailed the random selection of five ostrich chicks every day from hatching up to seven days, with the subsequent measurement of the yolk content parameters. Amino acids and fatty acids were only analysed on days one, three, five and seven.
Linear regression functions were fitted to weight and yolk-variables over the period of seven days post-hatching to quantify changes over time.
Amino acid and fatty acid composition was subjected to one-way analysis of variance using SAS version 9.2 (SAS, 2000). Levene‘s test was performed to test for homogeneity of daily analysis of variance (Levene, 1960). The Shapiro-Wilk test was performed to test for non-normality (Shapiro & Wilk, 1965). Student‘s t-Least Significant Difference was calculated at the 5% confidence level to compare means for age (days) (Ott, 1998).
The experimental design for the second trial also entailed random selection of chicks with body weight and yolk observations made on six chicks every second day from hatching up to sixteen days. Linear regression functions were fitted on body weight and yolk-variables over the period of sixteen days post-hatching to quantify change over time.

Results and Discussion

Starved chicks trial

There was no difference in the hatching weight between the different chicks (P = 0.56) (Table 1). The average hatching weight was much lower than reported in other ostrich chick studies which ranged from 1.4-1.6 kg (Keffen & Jarvis, 1984; Mushi et al., 2004). There was a decrease (P<0.05) in slaughter weight from one to seven days post-hatching (Table 1). The chicks lost an average of 31.3 g body weight over the first seven days post-hatching (Figure 1). This was expected as the chicks did not receive any feed and utilised 44% of their yolk over the trial period

Chapter 1: General introduction 
Hypotheses
References
Part 1: Yolk utilisation and the development of the small intestine of ostrich chicks 
Chapter 2: The composition of egg yolk absorbed by starved ostrich (Struthio camelus L.) chicks from one to seven days post-hatching and for ostrich (Struthio camelus L.) chicks from one to sixteen days posthatching on a pre-starter broiler diet
Introduction
Materials and Methods
Results and Discussion
Conclusion
References
Chapter 3: Enzyme activity in the small intestine of ostrich (Struthio camelus L.) chicks from two to sixteen days post-hatching on a pre-starter broiler diet
Introduction
Materials and Methods
Results and Discussion
Conclusion
References
Chapter 4: A histological and morphometric study of the small intestine of ostrich (Struthio camelus L.) chicks from two to sixteen days post-hatching on a pre-starter broiler diet
Introduction
Materials and Methods
Results
Discussion
Conclusion
References
Part 2: Influence of various pre-starter diets on growth and the development of the small intestine of ostrich chicks 
Chapter 5: A growth and digestibility study of ostrich (Struthio camelus L.) chicks on eight different prestarter diets
Introduction
Materials and Methods
Results and Discussion
Conclusion
References
Chapter 6: Enzyme activity in the small intestine of ostrich (Struthio camelus L.) chicks on eight different pre-starter diets
Introduction
Materials and Methods
Results
Discussion
Conclusion
References
Chapter 7: A histological and morphometric study of the intestinal tract of ostrich (Struthio camelus L.) chicks on eight different pre-starter diets
Introduction
Materials and Methods
Results
Discussion
Conclusion
References
Chapter 8: General conclusion and future perspective 
Recommendations
References
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