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Effect of feeding graded levels of cottonseed meal on goat erythrocyte membrane osmotic fragility

G H M Matondi, E Masama*, I D T Mpofu and F F Muronzi

Department of Animal Science, Faculty of Agriculture, University of Zimbabwe, P. O Box. MP 167, Mt Pleasant, Harare, Zimbabwe
*Department of Animal Science and Technology, Bahir Dar University, P.O. Box 79, Bahir Dar, Ethiopia.


The effect of feeding graded levels of cottonseed meal in goat rations on erythrocyte membrane osmotic fragility was examined. Goats (n=16) were randomly assigned to four diets according to age groups. The diets were isonitrogenous and isoenergetic and contained 0, 8, 16, and 24% cottonseed meal.  Blood samples were collected at the start of the experiment and after every two weeks for the next six weeks.


Erythrocyte membrane osmotic fragility increased (P<0.001) in diets containing cottonseed meal. Erythrocyte membrane osmotic fragility was greater (P<0.001) in high gossypol containing diets, 24 >16 >8% cottonseed meal (CSM) and increased with exposure time.  Erythrocyte membrane osmotic fragility differed among diets and increased with increasing gossypol intake. Erythrocyte fragility for diets with 0% and 8% CSM was less than 10% and ranged between 14.6 and 26.43% for diets with 16 and 18% CSM in week six. Erythrocyte fragility was less than 5% for all the diets in week one. Gossypol toxicity was not observed. The absence of gossypol toxicity in these animals was attributed to the low free gossypol content of the diets, reduced intake with time and to the detoxification of free gossypol in the rumen.


Erythrocyte fragility can be used to assess effect of consumption of diets with gossypol by goats.

Key words: cottonseed meal, erythrocyte osmotic fragility, goats, gossypol


In Zimbabwe, cottonseed meal is a major ingredient used in stockfeeds manufacturing. It is cheaper, compared to soyabean meal, and has less competing uses. However, the presence of antinutritional factors, especially gossypol limits its use as a feed ingredient. Gossypol is a yellow polyphenolic compound found primarily in the pigment glands of the cotton plant (highly concentrated in seeds) and it exists in both the bound and the free forms. The free form is biologically active. Dietary gossypol from cottonseed products has been shown to cause haematological changes in ruminants (Lindsey et al 1980). Increase in erythrocyte fragility is one haematological change that occurs in animals consuming diets with gossypol. Gossypol toxicosis causes erythrocyte membrane fragility Gray et al (1993) and Mena et al (2004). These haematological changes affect animal health and performance.


Lindsey et al (1980) indicated that many effects of gossypol toxicity occur in animals and the major effects include alteration in normal erythrocyte structure and decreased haemoglobin. Calhoun et al (1990a) reported erythrocyte fragility in feeder lambs and Gray et al (1993) in heifers given moderate amounts of products containing free gossypol. Erythrocyte fragility is a measure of the ability of the erythrocyte membrane to resist osmotic stress when erythrocytes are incubated in different salt concentrations. According to Velasquez-Pereira et al (1998) erythrocyte fragility is a very sensitive indicator of systemic gossypol status because it is evident shortly after gossypol consumption has started. They also noted that the mechanism of gossypol effect on the erythrocyte is not well understood. Calhoun et al (1990b) noted that increased red blood cell fragility is a good indicator of gossypol intoxication in goats.


The effect of gossypol on erythrocyte fragility can be used to measure the safe feeding levels of cottonseed products to animals. Velasquez-Pereira et al (1998) proposed that erythrocyte fragility can be used as a sensitive indicator of systemic gossypol status. The level of free gossypol in cottonseed meal that can be tolerated by goats has not been determined in Zimbabwe since many studies have been done with either sheep or cattle. The objective of this study was to determine the effect of feeding diets with graded levels of cottonseed meal (hence different free gossypol content per diet) on erythrocyte osmotic fragility of indigenous goats.


Materials and methods


Chemical analysis


Feed and refusals were ground to pass through a 1mm screen using a Wiley mill, before chemical analyses. Dietary ingredients and diet samples were analysed for DM, CP, EE and ash content using the Official Methods of Analysis of the Association of Official Analytical Chemists (AOAC) (1990). Calcium and phosphorus content of the materials was determined by wet chemistry as stipulated by the AOAC (1990). Determinations of the fibre fraction, neutral detergent fibre (NDF) and acid detergent fibre (ADF) were done as described by Goering and Van Soest (1970). Each analysis was done in triplicate. Oven dried feed refusals and faeces were also analysed for dry matter (DM) and organic matter (OM) (AOAC 1990). Free gossypol content in feedstuffs and diets was determined by aniline reaction procedures according to the American Oil Chemists Society methods Ba 7-58 (AOCS 1988).


Feed ingredients and ration formulation


Treatment diets were formulated using maize meal, soyabean meal (SBM), cottonseed meal (CSM), feed-grade limestone, mono-calcium phosphate (MCP), vitamin-mineral premix and wheat straw which was added ad lib as a roughage source. The chemical composition of the major dietary ingredients is shown in Table 1 while Table 2 shows the ingredients and chemical composition of treatment diets (DM basis).  

Table 1.  Chemical composition of dietary ingredients on dry matter (DM) basis


Parameter, %









Free gossypol

Cottonseed meal










Full fat soyabean meal










Maize grain (crushed)










Wheat straw










Table 2.  Chemical composition of experimental diets 


Parameter, %

*Free gossypol, mg/kg










0% CSM










8% CSM










16% CSM










24% CSM










*Calculated values

Ingredients were manually mixed and packaged in 50 kg bags. Representative samples of each treatment diet were collected and analysed for crude protein (CP), dry matter (DM), ether extractives (EE), acid detergent fibre (ADF), neutral detergent fibre (NDF), calcium, phosphorus, ash and free gossypol.


Experimental design


Sixteen indigenous female Mashona goats were placed in four age groups of < 6, 6-12, 12-18 and > 18 months. Age was estimated by dentition because of the absence of breeding records. Animals were randomly assigned to four treatment diets containing 0, 8, 16, and 24% CSM in a completely randomised design with four age groups. The control diet contained soyabean meal (SBM) as the main protein source. The free gossypol content in each group was calculated by multiplying the free gossypol content of CSM by the inclusion level of the CSM in each diet. This was done because determination of free gossypol in complete diets using the aniline reactions is unsatisfactory (Risco and Chase 1997).


Animal housing and feeding


All animals were dewormed and vaccinated against blue tongue, pulpy kidney and black leg using a broad spectrum vaccine before the start of the experiment. Each animal was housed in an individual metabolism crate, measuring 0.6 x 0.75 x 1.0 m and raised 0.5 m above the floor in the Bioassay Laboratory with normal lighting, Department of Animal Science at the University of Zimbabwe. Each metabolism crate was fitted with a feeding trough and an automatic nipple drinker. Animals were fed twice a day, around 0800 and around 1200 hours. Concentrate contributed 30% and wheat straw 70% of the feed given to goats amounting to 3% of body weight. 


Blood collection


Blood samples were collected two hours after the morning feeding. Blood samples (10 ml) were collected at the beginning of the study and every two weeks for six weeks by jugular venipuncture using 18-gauge needles and 10 ml syringes, into blood collection tubes containing ethylenediaminetetraacetic acid (EDTA), an anticoagulant. A fresh set of needles and disposable syringes were used for each animal as a precaution against possible transfer of infection from one animal to another. The needles were removed so as to prevent mechanical haemolysis Chivandi (2000) when the blood was being transferred to the collecting tubes. The tubes into which the blood was put were labeled with animal identity, treatment diet number and date of bleeding.


Estimation of osmotic fragility of erythrocytes


Erythrocyte membrane osmotic fragility was estimated in 0.65% saline, which was prepared from 10% NaCl solution (stock solution) and included Na2HPO4 as described by Risco et al (1993). Animals were observed daily for any signs of illness during the feeding period.


Statistical analysis


Data relating to erythrocyte membrane osmotic fragility was analysed by GLM procedures of SAS program SAS (1996). Repeated measures of erythrocyte fragility was evaluated by ANOVA for repeated measures using the PROC MIXED procedure of SAS, Littell et al (1998) including the effects of diet, age of animal, day of blood collection and interactions between diet and day of blood collection, age by diet and day of blood collection age.


Results and discussion


Effect of free gossypol in cottonseed meal on health of animals


No goats had visible signs of gossypol toxicity, which include dyspnoea, anorexia, weakness and sudden death during the 42-day study. This was despite the fact that the diets which contained CSM increased erythrocyte fragility (P<0.001, Table 3).

Table 3.  LS means for weekly erythrocyte fragility (%) by diet treatment and the standard error of LS means (S.E.M.)










0% CSM








8% CSM








16% CSM








24% CSM








abcdWithin rows means with different superscripts are different (P<0.05)

Menges (1991) indicated that increased red blood cell fragility is a good indicator of gossypol intoxication in goats and in bulls Wyse et al (1991) and in dairy cows Lindsey et al (1980). These findings also agree with those of Mena et al (2004) who observed that feeding lactating dairy cows high free and total gossypol diets for 42 days resulted in no signs of gossypol toxicosis. Toxicity effects are expected when high concentrations of free gossypol are rationed, ruminants have the capacity to detoxify large amounts of free gossypol by binding it to soluble proteins in the rumen Reiser and Fu (1962). This could have contributed to the lack of visible signs of gossypol toxicity in this study. Animals might have avoided toxic effects by adjusting to lower intakes.


The increase in erythrocyte fragility in animals that consumed a gossypol-containing diet is an indicator of gossypol intake. In this experiment, the effect of gossypol on erythrocyte fragility was not accompanied by any other clinical signs of gossypol toxicity.


Effect of dietary treatment and exposure time on erythrocyte osmotic fragility


Time of exposure to dietary treatments and their interaction with diets increased erythrocyte fragility of red blood cells (P<0.001). Cottonseed meal inclusion in diets increased erythrocyte fragility (P<0.001, Table 3 and Figure 1) from week two of feeding.

Figure 1.  
Effect of dietary treatment and exposure time (week)
on erythrocyte osmotic fragility of red blood cells

This result agrees with observations by Velasquez-Perreira et al (1999) that the effect of gossypol on animal performance is cumulative. Consumption of gossypol increased erythrocyte fragility (P<0.05) in the order 0, < 8, < 16, < 24% CSM throughout the study period. These results agree with observations by Mena et al (2004) who observed that in cows given high free gossypol, erythrocyte fragility increased. These findings concur with results from other studies that have suggested that erythrocyte fragility is sensitive to amount of gossypol consumed Lindsey et al (1980); Risco et al (1993) and Velasquez-Pereira et al (1998). Reyes et al (1984) observed that gossypol binds strongly to lipid bilayers and induces an electrical conductance that is accompanied by an increase in proton permeability. This affects the fluidity of membranes. They also demonstrated that gossypol caused an increase in diffusion in lipid membranes in the presence of NaCl, which could explain the increase in erythrocyte fragility in this study. Another study with bovines by de Peyster et al (1986) showed an increase in membrane permeability because of gossypol treatment.


De Peyster et al (1986) suggested that degeneration of cell membranes at all levels of organisation may exist with acute and prolonged exposure to gossypol in vivo. This suggestion is supported by our results in that erythrocyte fragility increased significantly as exposure time to diets with gossypol increased from week one to week six.


The effect of gossypol on erythrocyte fragility also exhibited a dose (gossypol concentration) -time dependency. Erythrocyte fragility increased (P<0.001, Table 3 and Figure 2) with time and with an increase in the concentration of free gossypol in the diet.

Figure 2.
 Effect of age of animal and diet on erythrocyte fragility

Velasquez-Pereira et al (1998) indicated that the effect of gossypol is both dose and time-dependent and that multiple oral doses gradually increase the amount of the toxicant that is retained in the body.  The effect of gossypol on erythrocyte fragility has been reported in many animal species including cattle, sheep, goats and pigs Menges (1991) and Gray et al 1993). Increase in erythrocyte membrane osmotic fragility is a sign of gossypol intoxication of goats in this study.


Effect of interaction between dietary treatment and animal age on erythrocyte fragility


Animal age and diet had an effect on erythrocyte fragility (P<0.001, Table 4 and Figure 2).

Table 4.  Effect of dietary treatment and age of animal on erythrocyte fragility by age of animals and the standard error of ls means (S.E.M)

Animal age

Cottonseed meal, %






>18 months






12-18 months






6-12 months






<6 months






abcdWithin column Lsmeans with different superscripts are different (P<0.05) 1234Within row means with different superscripts are different.

Erythrocyte membrane osmotic fragility increased with a decrease in the age of the animal, that is the younger the animals, the greater their susceptibility to gossypol toxicity. Risco et al (1992) noted that calves with undeveloped rumens (prefunctional ruminants) cannot efficiently detoxify gossypol, hence response between mature ruminants and immature ruminants would be expected to be different. In this experiment, older and heavier goats (more than 18 months), recorded significantly lower erythrocyte fragility (P<0.05) than animals in the weaner and growing age groups (less than 12 months of age). This could have been due to the size of the rumen which increased the capacity to detoxify gossypol. Dietary treatments had different effects (P<0.05) in all the four age groups with fragility of red blood cells being correspondingly higher in young and growing animals eating diets with more cottonseed meal. These results agree with earlier observations that ruminants are able to detoxify gossypol in the rumen (Risco et al 1992).


Bound gossypol may be converted to the free gossypol in the gastrointestinal tract Baliga and Lyman (1957). Since most of the gossypol in CSM is in the bound form some of it might have been converted to free form during digestion Blackwelder et al (1998) and Noftsger et al (2000) which is biologically active hence the change in erythrocyte fragility of red blood cells.



The authors are grateful to Regional Universities Forum for Capacity Building in Agriculture (RUFORUM) for funding this research.




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Received 2 August 2007; Accepted 25 August 2007; Published 1 November 2007

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