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Regular Article |
a Sección de Recursos Cinegéticos, IDR, Universidad de Castilla-La Mancha, 02071 Albacete, Spain
b Departamento de Ciencia y Tecnología Agroforestal, ETSIA, Universidad de Castilla-La Mancha, 02071 Albacete, Spain
c Instituto de Investigación en Recursos Cinegéticos (CSIC-UCLM-JCCLM), Sección Albacete, Universidad de Castilla-La Mancha, 02071 Albacete, Spain
d Departamento de Química Inorgánica, Orgánica y Bioquímica, Facultad de Medicina, Universidad de Castilla-La Mancha, 02071 Albacete, Spain
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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early development, environment, immunology, lactation, stress
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Increasing evidence indicates that immune defense is compromised by limiting access to resources, such as energy or protein [5–8]. It is expected that this should be particularly the case during lactation. Although, as mentioned above, milk in ruminants is produced from food taken on a daily basis, it is well known that lactating cows can assume a negative protein balance during early lactation. However, the ability to mobilize tissue protein for milk production is small relative to the ability to mobilize fat. Protein body reserves are below 5% of total body protein in most mammals, and in cows, they could not support more than 20% of daily milk production for longer than 14 days [9]. In contrast, when protein is not severely limiting but metabolizable energy intake is reduced, fat reserves are mobilized greatly to maximize milk production [1]. This mobilization of fat can produce a compensatory response in deer lasting up to 4 wk under a sharp reduction in food availability [10].
Because newborns have impaired ability to produce antibodies and are more dependent on passively transferred immunity from the mother's milk than adults are, and because intestinal epithelium loses its ability to absorb intact macromolecules around 24 h after birth, colostrum is the main source of antibodies and immunity proteins for newborn calves [11]. Immunoglobulins (Ig) in colostrum account for 70–80% of their protein content and are the result of selective accumulation from plasma Ig that starts several weeks before parturition [12]. Thus, it is unlikely that food restriction after calving would affect Ig content in colostrum. However, there are controversial findings suggesting that food restriction may affect calf absorption of such Ig [11]. Protein from mature milk also contains 1–2% of Ig [12] that appears to retain immunological activity in the intestine of the recipients [13]. At least content of the most important Ig in milk, IgGs, is related to the serum levels of IgG in cattle mothers [14, 15], whereas mortality rates of calves with low serum IgG are attributed to lack of IgG intake with milk [11].
Although, as mentioned above, most research on passive immunity transfer by milk intake has focused on Ig levels, humoral immunity is only one of the components of a complex system, which includes cell-mediated and other responses. Some authors have warned that single-variable studies suffer from an increased risk of erroneously concluding that no relationship exists between immunocompetence and life-history decisions (e.g., to reproduce, to grow [16]), although significant results obtained in those studies are regarded as reliable evidence for such a relationship.
The aim of this study was to assess the effect of nutrition stress on immunocompetence costs by examining potential relationships between Ig levels in blood; serum indices of body condition, such as albumin; lactation variables, such as milk production and composition; and weight trends in hinds and calves. Because nutrient stress started at lactation, the potential effects may simulate drastic reductions of available food occurring precisely at lactation rather than density-dependent reductions of nutrients that would affect also gestation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Twelve red deer hinds of the Iberian subspecies (Cervus elaphus hispanicus) were used in this study. The mean age of the food-restricted hinds during lactation was 5.0 ± 1.55 yr (mean ± SD), whereas the control group had a mean age of 5.5 ± 1.64 yr. The first group was composed by four male and two female calves, whereas all the calves in the control group were males.
The control group was maintained in a 10 000 m2 open-door enclosure on an irrigated pasture including tall fescue, Festuca arundinacea (52.4%); cocksfoot, Dactylis glomerata (28.6%); lucerne, Medicago sativa (14.3%); and white clover, Trifolium repens (4.8%). In addition, this group had access to 1 kg/day/individual of diet based on suggestions by Brelurut et al. [17], using barley straw and hay from barley, alfalfa, oat, and sweet beetroot (16% crude protein). The food-restricted group was maintained in a similar enclosure without pasture and had access to 1.5 kg of the previously mentioned diet (about 50–60% of caloric intake suggested during lactation). Average hind weights at the beginning of the experiment (at calving) were 101.2 ± 14.2 and 103.7 ± 8.3 kg (mean ± SD) for the experimental and control group, respectively.
To increase definition in early lactation, when milk production changes most as lactation proceeds, hinds were milked on Weeks 2, 3, 4, and 6 and then every 4 wk up to Week 18 in this and another simultaneous experiment involving two additional groups. However, for ethical reasons based on the poor body condition of the group on low caloric intake, in this study, food restriction in the experimental group ended on Week 10. Blood samples were taken from the right jugular vein at the time of milking. To keep social conditions constant throughout the experiment, weaning was enforced simultaneously in all hinds within a group. Before each milking, hinds were separated from their calves for 6 h (0800–1400 h) in a deer-handling facility. For ethical reasons fully explained in [18], no milking prior to this isolation period was conducted to dry out the udder. Milking was carried out, under anesthesia, with a milking machine set up to 50/50 massage/milking ratio and 44 kPa of vacuum. Machine milking was performed in 30–60 sec, and after this, the hind was hand milked to collect the remaining milk. Daily milk production was estimated to be four times the volume collected in each milking. Milking frequency was reduced to the minimum considered essential to prevent stress and potential damaging effects of the anesthesia, which was achieved using a low-dose combination of xylazine (0.5 mg/kg body mass) and ketamine (1 mg/kg) delivered by i.v. injection. After anesthesia was induced, 10 IU of oxytocin were injected in the right jugular vein 1 min before starting milking. After milking was finished, anesthesia was reversed with an injection of yohimbine (0.25 mg/kg body mass). Hinds were weighed weekly on a ±50 g electronic balance, whereas calves up to 35 kg body mass were weighed in a ±5 g balance and in the same balance as their mothers thereafter.
Serum protein analysis was done by using a Paragon electrophoresis kit (Paragon SPE, Beckman Instruments, Brea, CA). Fractions were subsequently quantified using an Appraise densitometer (Beckman Instruments). Total serum proteins (TSP) were assessed with an Atago SPR-T2 refractometer (Atago Co., Ltd., Tokyo, Japan).
In addition, two 30-ml samples of milk (replicates) were collected for chemical analysis. Milk analyses were carried out in an automatic milk analyzer as described in [19].
Statistical Analysis
A one-way ANOVA tested differences between groups in hind age and weight variables, those of the calf, serum proteins, and milk production and composition. Pearson correlations (bilateral) tested the relationships between serum proteins, lactation, and weight variables of the hinds and calves. Student t-tests were used to study within-week differences for each of the variables monitored during lactation. Because the behavior of most variables in another part of the study showed two phases in the physiological response (from birth to the fourth week and from Week 4 until the end of the experiment, i.e., the 10th week), we also analyzed these two phases separately.
Following statistical guidelines in top-ranking journals on milk and animal science [20, 21] and as a result of the small sample size involved, marginally significant tests (P < 0.1) are reported by their exact probability rather than as P > 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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No difference between groups of hinds was found in TSP or serum Ig except when the percent of Ig relative to TSP (PIg) was considered for the overall lactation or for the period after Week 4 (Table 1). In contrast, calves under low caloric intake showed higher values for both total levels of Ig (TIg) and PIg, but not for TSP, during all periods (Table 1).
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There was a similar lack of significant difference between groups in serum albumin levels (both in absolute values [TA] and percent relative to TSP [PA]) and albumin-to-globulin ratio (A:G), with only marginal values of significance for both PA and A:G in calves up to Week 4 and in hinds after this week (data not shown).
Nutritionally stressed hinds did not appear to be greatly affected in humoral immunocompetence indicators or serum indices of body condition. Only TSP correlated positively with both hind weight change (R = 0.62, P = 0.03) and percentage of weight change during lactation (R = 0.63, P = 0.028). Hinds giving birth to heavier calves showed significant or marginally significant decreases in PA and A:G (calf body weight [CBW] vs. PA or A:G, R = -0.71; R = -0.70, P < 0.05, respectively), whereas hinds producing more milk (total milk yield [TMY]) or its nutrients (total fat yield [TFY], total protein yield [TPY], total lactose yield [TLY]) showed lower PA in late lactation (PA vs. TMY, TPY, or TLY, R =-0.52, P = 0.081; R = -0.51, P = 0.093; R = -0.52, P = 0.081, respectively).
In contrast, low caloric intake from their mothers clearly affected calf humoral immunity. Calves showed a negative correlation between calf gains or percentage of gains and both PIg during the entire lactation or between both gain variables and TIg or PIg after the fourth week (Table 2). Similarly, total milk production and that of milk constituents also yielded a negative correlation with both PIg for total lactation and with PIg or TIg after Week 4.
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Regarding serum indices of body condition, calves showed again strong positive correlations between the three indices and gains (absolute or percent) up to Week 4 and between these and PA or A:G for the entire lactation period (Table 3). Correlations between all albumin indices and production of milk or production of milk nutrients were positive and significant or marginally significant up to Week 4 and marginally significant between PA or A:G and milk production or most of its nutrients during the entire lactation period.
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The time course of serum immunocompetence, as determined by levels of immunoglobulins, shows a similar difference between hinds and calves (Figs. 1–3). Within-week differences in TSP between groups did not show any clear trend (Fig. 1). In contrast with hinds, calves in the nutritionally stressed group showed significantly higher TIg (Fig. 2) and PIg (Fig. 3) values than calves in the standard diet group for most weeks. The differences were larger and more consistent (including also Week 10) in TIg.
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Correlations Within Groups
Despite the small sample size (n = 6), nutritionally stressed hinds showed a strong inverse relationship between serum condition indices and calf birth weight for the entire lactation period (CBW vs. PA, TA, and A:G [R = -0.97, P < 0.001; R = -0.90, P = 0.014; and R = -0.96, P = 0.002, respectively]) for the period up to Week 4 (very similar coefficients) and for the period after this week to Week 10 (CBW vs. percentage of albumin R = -0.92, P = 009; CBW vs. A:G, R = -0.92, P < 0.01). No such relationship was found for serum Ig.
Hinds producing more milk and nutrients increased calf weight gains both in total values and percentage (data not shown). In addition, they showed higher serum Ig levels both up to Week 4 (Table 4 for this and following comparisons) and for the entire lactation period but not for the period after Week 4. There was also a positive correlation between milk or milk nutrient produced and hind PIg or TIg up to Week 4 and between hind PIg and nutrients produced for the entire lactation period but not for the period after Week 4. Hinds producing milk with greater protein content also had higher Ig values up to Week 4 (milk protein content vs. hind TIg, R = 0.95, P = 003), but no other correlation was significant for any other milk component at any lactation stage. Hind levels of TA and TIg showed a positive correlation after Week 4 (R = 0.910, P = 0.12) and a marginally significant one for the entire lactation period (TIg vs. A:G ratio, R = 0.754, P = 0.083).
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In contrast, hinds under standard nutrition conditions showed negative correlations between Ig content vs. weight gains of their calves for the entire lactation (Table 4) and between Ig vs. yield of milk or its nutrients up to Week 4. Albumin levels, however, maintained a negative correlation with calf weight gain (hind TA vs. calf weight gain up to Week 4, R = -0.932, P = 0.007).
Regarding nutritionally stressed calves, a strong positive correlation was found between weight gains (total and percentage) and serum indices of body condition both for the entire lactation period (see Table 5 for this and following comparisons) and up to Week 4 but not after Week 4. Serum indices of calf body condition, particularly TA, showed correlation with milk and nutrient production up to Week 4 but not at any other stage.
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Strikingly, a positive correlation was observed between calf serum Ig and milk nutrient production up to Week 4 (see Table 6 for this and the following comparisons). Protein content correlated with both calf TIg and PIg up to Week 4 (R = 0.82, P = 0.046; R = 0.78, P = 0.66, respectively), PIg correlated with protein to fat ratio (R = 0.87, P = 0.023), and there was a marginally significant correlation with milk fat content (PIg vs. fat content, R = -0.74, P = 0.90) for the same period but not at any other stage. Correlations with calf gains for this period were positive and around 0.7, but they failed to reach significance. In contrast, correlations became negative after this week, although only the correlation between milk protein yield and calf PIg reached significance. This inverse trend may be the explanation for the absence of significant correlation between these variables for the entire lactation period.
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Similarly, within the group on a standard diet, there was a similar positive correlation between calf weight gains and calf albumin variables (Table 5) and between TA and yield of milk and its nutrients for the entire lactation. However, there was a negative correlation between percentage of body weight gained by the calf and its PIg (Table 6).
Finally, the only significant relationship between serum indices of body condition and the levels of Ig was found in the group on a standard diet after Week 4: the greater the calf PA or A:G, the smaller the calf TIg (PA vs. PIg or TIg, R = -0.853, P = 0.031; R = -0.774, P = 0.071; A:G, R = -0.831, P = 0.040; R = -0.731, P = 0.099, respectively).
Weight Differences after the Experiment
Hinds under nutritional stress were lighter at the end of the 10th week than those under a standard diet (88.3 ± 4.6 vs. 103.7 ± 3.6 kg, respectively, P < 0.001). Despite an enforced weaning of the nutritionally stressed hinds (not of the calves in the control group that continued lactation until Week 14) and despite food being provided ad libitum to recover their body condition, marginal differences were maintained up to the time of the following rut in mid-September (P = 0.061; correlation coefficient of hind weight at weaning vs. weight at rut, R = 0.98, P < 0.001). Three of the calves under nutritional stress (those with lower weights at weaning) died before their first winter of life. Including the rest of the data, the weight of the calves at weaning correlated with that at the end of autumn-onset of winter (mid-November, R = 0.93, P < 0.001) and at 12 mo (R = 0.79, P = 0.012).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Overall comparisons between groups appear to support the second hypothesis, as mothers of calves under nutritional stress produced less milk and nutrients and calves showed smaller weight gains but both total and relative Ig levels remained high throughout lactation. Correlations between Ig levels and other variables, however, exhibited a more complicated pattern. Levels of Ig did not show a general correlation pattern with either weight gains, milk nutrient production, or albumin levels, which suggests either no effect, a subtle effect (thus needing a greater sample size), or inverse patterns within each group. Only during the period after Week 4 Ig levels showed a general negative correlation with weight gains and milk nutrient supply. Previous unpublished results from our group showed that, after Week 4, hinds lost their ability to counterbalance low food intake with their own body reserves and nutrition stress exerted a sharp effect both on calves and hinds. Thus, when hinds lose their ability to compensate a reduction in food intake, Ig levels in calves increase, which may be related to a poorer body condition and increased susceptibility to infection or attack by parasites. This is further suggested by the significant negative correlations precisely during this period between Ig levels and serum body condition indices (albumin or A:G ratio) in calves within the group on a standard nutrition diet.
Both for normal or low nutritional conditions, during compensatory period up to Week 4 but also during the entire lactation, albumin and other serum indices of body condition did correlate positively with calf weight gains and milk or milk nutrient production. However, an inverse pattern within each group appears to be the reason for the lack of significant correlation with Ig levels. In calves under standard nutrition conditions, the lower the level of Ig, the better the body condition as measured by albumin levels and the greater the growth rate. This suggests that Ig levels might reflect a low level of ongoing defense against infection or parasite attack, which is paired with a good body condition. However, in calves under nutritional stress, Ig level was positively correlated with milk protein content and the production of milk and its nutrients. The correlation of such production with growth and the correlation of total or relative weight gains or milk nutrient production with albumin levels suggest that Ig levels did not reflect poor body condition and high infection level but a shift of nutrient intake toward the production of Ig. Davis [22] proposed a mechanism by which, under nutritional stress, growth hormone would shift the flow of nutrients from muscles toward tissues of higher priority in such circumstances, such as the immune system. A second mechanism may have acted also in this case because prolactin under stress would also stimulate the immune system [22]. This hormone is known to increase under nutrition stress during lactation, at least in hinds [23]. Thus, in calves under nutritional stress and at least during the compensatory period up to Week 4, Ig levels would correlate with their ability to fight infection.
The double pattern found under different planes of nutrition might be the result of a sequential effect of nutrient reduction on the immune system. The first line of defense against pathogens is innate immunity: epithelial barriers, mucus secretion, antibodies secreted into the digestive tract and mucoses, and others [24]. Only after this fails do pathogens need to be fought using the adaptive humoral system constituted by Ig. If food restriction affects first innate immunity and subsequently humoral immunity, then pathogens will only affect those calves under standard nutrition that have a poorer body condition. In contrast, once overcoming the first barrier of defense in calves under nutritional stress, calves with more resources will be better able to fight off pathogens, thus showing a higher level of Ig.
To demonstrate whether the previous mechanism is true, we need to monitor parasite or pathogen load. Health management regime and ethical reasons impose antiparasite treatments, and no parasite was detected in feces and other controls. If Ig reflects infection level, however, it might do so in response to opportunistic infections by protozoans, bacteria, or viruses. Scanning for all of them in blood or other body samples to correlate their load with Ig level appears to be a huge task. There is indirect evidence linking albumin and parasite load under different nutrition and body condition sets. In studies involving wild deer, there is evidence of a higher parasite load in deer under poorer body condition and lower albumin and A:G ratio [25]. An inverse relationship between albumin and parasite load in free-ranging lactation would support the double pattern shown here.
Although the present study does not allow us to assess the potential cause of a decline of the barriers of defense against infection, a decrease in Ig enteric levels as a result of a reduction in milk contribution might be very likely. Most studies on passive immunity have focused on colostrum, which contains a high proportion of its protein in the form of Ig and other immune proteins [11]. However, ordinary milk contains 1–2% Ig [12], mostly IgG [14, 15] and also other immunoglobulin types such as IgM and IgA [12]. Some studies have found that at least about 19% of ingested IgG and IgM maintains its immunological activity in the ileum of healthy human adults [13]. Thus, milk Ig may contribute to deterrence of enteric infections in the calf, and a reduction in milk intake might result in a greater infection rate for calves under nutrition stress, thus forcing them to use their nutrient reserves to produce serum Ig. The level of IgA might be particularly important, as it is secreted outside the epithelium and into the digestive tract and mouth to fight pathogens [24].
Hinds also showed a similar pattern in correlations regarding Ig levels. Whereas hinds under nutritional stress had higher Ig levels the greater the weight gain of their calves and the greater their milk and nutrient production or protein content up to Week 4, those under standard nutrition showed a negative relationship with these variables.
Diverting nutrients to immunity to secure survival might have consequences for future reproduction for both calf and hind. Although no data is available in red deer, mortality rates in cattle calves with serum IgG concentrations lower than standard levels were over twice those of calves with such levels [11]. This might prompt calves to trade-off immunity for growth to ensure survival. However, weight at the end of weaning showed a high correlation with that at the onset of winter and at 12 mo, whereas weight at the end of the first year of life in red deer is related to adult weight and fighting ability, at least in males [26]. Thus, diversion of nutrients away from growth may impact chances of holding a harem and therefore future reproduction.
A similar effect may occur in the mothers. Again, the data on cattle shows that about 50% of deaths of cattle calves with low serum IgG concentrations were attributed to lack of IgG intake [11]. The mobilization of body reserves to keep milk production under nutritional stress appears to have resulted in increased serum Ig, but at the expense of losing hind body weight. Despite an earlier ad libitum diet to recover from lactation, hinds in the nutrition stress group had overall a lower weight at mating than the standard diet group did, whereas there was a high correlation between individual weights at both stages. As fertility in red deer and other ruminants is closely related to female body weight [3], the investment in greater milk production appears to have produced better immunocompetence in calves, but at the expense of lower reproduction chances in the following season.
To our knowledge, this is not only the first study assessing how nutrient availability affects the trade-off between immunity and lactation in a wild mammal but also in the most studied case of cattle. In addition to their biological and ecological consequences, there might be a methodological warning to draw from this study. Many studies involving immunocompetence rest on the assumption that the monitored indices of the immune system (Ig in our case) maintain a simple relationship with other variables such as body weight, dominance, or milk production, even if the groups compared had a different population density, nutrient availability, and so on. However, researchers should note that body variables might bear a different relationship with the immunocompetence index used in different environmental conditions.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX: 34 967 599238; landete{at}cita-ab.uclm.es
Received: 13 February 2002.
First decision: 4 March 2002.
Accepted: 25 May 2002.
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