Praca oryginalna Original paper
Managed dairy cows, especially of the Holstein--Friesian breed, are currently able to produce extremely large amounts of milk; however, this is increasingly often accompanied by disadvantageous side-effects caused by the increase in negative energy balance (NEB) observed during the final period of pregnancy and initial period of lactation. More precisely, NEB commences about 3 weeks before expected calving, is highest about 3 weeks after calving, and lasts usually up to the 6-7th week, and in extreme cases even up to
the 11-12th week of lactation (15). Moreover, NEB in
the given time-period is an evolutionarily determined physiological state, whereby excessive use of energy reserves deposited in the form of fat and other com-pound reserves leads to metabolic stress in a signifi-cant number of cows. As a consequence, an increased frequency of the occurrence of metabolic disorders, other diseases, disorders in milk production, and ferti-lity decrease are observed (4, 7, 9, 17, 22, 27). It was demonstrated that primiparous cows are more at risk
Evaluation of the possibilities for the use of blood
acetone concentration, body weight changes and
milk and reproduction performances as indicators
of dairy cow adaptation to metabolic stress during
the initial period of lactation
KRZYSZTOF A. BIA£OÑ, RYSZARD SKRZYPEK*
AdiFeed Sp. z o.o., Opaczewska Str. 43, 02-201 Warszawa, Poland
*Department of Cattle Breeding and Milk Production, Faculty of Animal Breeding and Biology, Poznañ University of Life Sciences, Wo³yñska Str. 33, 60-637 Poznañ, Poland
Bia³oñ K. A., Skrzypek R.
Evaluation of the possibilities for the use of blood acetone concentration, body weight changes and milk and reproduction performances as indicators of dairy cow adaptation to metabolic stress
during the initial period of lactation
Summary
The study was conducted on 124 Polish Holstein-Friesian primiparous cows of the black-white variety. The aim was to determine the relationship between the following indicators of adaptation to metabolic stress occurring in dairy cows in the early lactation stage due to negative energy balance: acetone concentration in blood (AcC) on days 5, 30, 60 and 90 after calving; body weight changes between days 5-60 after calving (BWC); milk yield (MY), fat content (FC) and protein content (PC) in milk, fat to protein ratio in milk (FPR) during the first 100 days of lactation; calving to conception interval (CC) and the number of AI services per conception (NSC). The data were analyzed using logistic regression. It was found that AcC positively influenced BWC, FC and FPR (OR > 1.0), and negatively PC (OR < 1.0), while the influence on MY was variable on subsequent dates of the examination, i.e. positive on days 5 and 30, and negative on days 60 and 90. In terms of the particular dates of AcC examinations, particularly large relationships were noted on the 60th day after
calving. It was also demonstrated that the relationship of AcC was reversible in the case of FC, and especially PC and FPR. With regards to fertility parameters, AcC and FPR influenced them to the highest degree on the 90th day after calving (OR from 6.4 to 8.9), and so did PC (OR between 0.3-0.4) and FC (OR between 1.1-1.9).
Concluding, among the tested traits, FPR is of the highest diagnostic value as an early indicator of metabolic stress, generally observed in dairy cows during the early lactation stage. A similar diagnostic value may be attributed to AcC; however, the possibilities of applying this parameter in practice are considerably lower.
from NEB and its negative consequences than multi-parous cows (10, 19, 33), and that detrimental biolo-gical effects of NEB pass from one lactation into the next (2). The consensus is that the recognition and solution of these problems is the biggest challenge for present-day dairy cattle breeders (22).
In this context, crucial to the issue is the ability to accurately assess energy balance during the initial lac-tation period and utilize the information thus obtained for the prevention of animal health problems using current management of the herd and genetic improve-ment. However, direct assessment is impossible in practice, since implementing a policy of control of individual feed intake on a herd-wise basis is imprac-ticable under production conditions. An alternative is indirect assessment using methods such as: analysis of production, reproduction, health and behavior para-meters; economic analysis; monitoring of changes in body weight and condition; and examination of bio-chemical parameters (2, 25, 27, 29). A promising option involves the application of the results of stan-dard milk performance testing, since such an approach simultaneously ensures a mass scale and regularity of the examinations, a lack of invasiveness, early dia-gnosis and very low costs (11, 18, 19, 25, 29).
The aim of the study was to determine the relation-ships between acetone concentration in blood, body weight changes and parameters of milk and reproduc-tion performance in primiparous cows of the Polish Holstein-Friesian breed. These characteristics were taken as adaptation indicators for metabolic stress that usually occurs during the early lactation period as a result of NEB. Since energy balance was not measured directly, the parameters of reproduction performance (calving to conception interval, number of AI services per conception) were used as the basis for an assess-ment of the early diagnostic value of other indices. In other words, it was accepted that among the analyzed parameters, only these ones accumulate the detrimental effects of NEB in the best manner, and are concurrently measurable relatively late in the cow reproduction--production cycle.
Material and methods
The study was conducted on 124 primiparous cows of the Polish Holstein-Friesian breed born, reared and managed in uniform conditions on the same farm, which is the Agri-cultural Experimental Farm D³oñ. The initial number of animals (7-8 weeks before the expected date of the first calving) was nearly 200, and only those of them that remained in the herd at least up to the second calving and were clinically healthy during this period were examined. During the first 305-day lactation they produced on average 6664 kg of milk, and the average length of their first calving interval was 384.1 days.
High-pregnant heifers and cows on the farm were main-tained in a tied system, in stalls of medium length littered
with straw. Irrespective of the season of the year, the animals used the extensive yard for at least 2 hours every day. They were fed in a traditional system, with two meals per day: in the morning (7:00 AM) and in the afternoon (4:00 PM). The rations were standardized according to the IZ PIB--INRA system (23). The basic ration for primiparous cows in lactation met the maintenance and production demands of animals with a body mass of 550 kg and an average daily milk yield of 19 kg containing 4.0% of fat and 3.1% of protein. The morning ration constituted 40% of the total daily intake, while the afternoon ration the remaining 60%. Leftovers were removed every day before the morning feeding. Lactating cows were additionally given a special complementary feed mixture, and its amount was strictly determined according to their level of milk production and lactation stage. The complementary mixture was fed with a premix with high-protein fodder material. The basic ration for primiparous cows in lactation was introduced for high-pregnant heifers three weeks before the expected calving. Basic roughages (maize silage and alfalfa silage) and components of a complementary feed mixture (ground maize, triticale, barley and wheat grains, and extracted rape meal) were fed at constant amounts throughout the period of the experiment.
Blood samples from the examined animals were collec-ted from the external jugular vein (v. jugularis externa) at 5, 30 (± 3), 60 (± 3) and 90 (± 3) days after calving. The samples were always collected at 10:00-11:00 AM, i.e. 3-4 hours after morning feeding. Serum was obtained during a period not exceeding one hour, after which it was cooled to a temperature of about +4°C. On the following day the concentration of acetone was determined using the gas chromatography method. On days 5 and 60 after calving, concurrently with blood sample collection, the cows were weighed, and on that basis average daily body weight changes were determined (BWC). The measurements were conducted at 12:00 AM 1:00 PM. Moreover, the research material consisted of production parameters during the first 100 days of lactation and reproduction parameters: milk yield (MY), fat content in milk (FC), protein content in milk (PC), FPR (FC/PC ratio), calving to conception interval (CC), number of AI services per conception (NSC).
The data were analyzed statistically using SAS software (30). First, arithmetic means and standard deviations were calculated using the MEANS procedure. The key calculations were conducted using logistic regression (the LOGISTIC procedure), according to the one-factor model containing an independent variable. The statistical method was selected; as it provides an odds ratio (OR) estimator it enables the analysis of relationships for dependent variables of binary character, as well as being very convenient in terms of the interpretation of the results of case-control medical and biological studies (5). For particular indicators the cows were divided into the following groups: AcC up to 0.4 mmol/L and above; BWC, milk performance features up to arithmetic mean and above; CC up to 90 days and above; NSC one treatment and above. The results were presented using OR statistics, which demonstrates the ratio of the probability of the occurrence of a specified state
(exposed or treatment group) to the probability of the lack of the occurrence of this state (non-exposed or control group), while the value of 1.0 is accepted in non-exposed group. An odds ratio higher than 1.0 implicates a positive relation-ship with a factor to which the exposed group is subjected, while an OR lower than 1.0 indicates a negative relation-ship. In the study, in cases when OR was lower than 1.0, a converse of the value estimated was also presented in the table in order to facilitate the comparison of the absolute size of relationships noted for all results of the calculations.
Results and discussion
Arithmetic means and standard deviations (in the brackets) for the analyzed traits were as follows: total AcC 0.39 mmol/L (± 0.12), BWC 445 g/day (± 734), MY 2650 kg (± 375), FC 4.00% (± 0.38), PC 3.05% (± 0.22), FPR 1.32 (± 0.16), CC 109.0 days (± 62.2) and NSC 1.90 (± 1.24). Analogical statistics for AcC at 5, 30, 60 and 90 days after calving were: 0.46 (± 0.30), 0.40 (± 0.24), 0.35 (± 0.14) and 0.35 (± 0.17) mmol/L, respectively. The level of AcC did not exceed 1.70 mmol/L on any of the dates of blood sampling. On the basis of the thresholds of 0.4 and 2.0 mmol/L accepted for subclinical and clinical ketosis, respecti-vely (1, 27), the results prove that to whatever degree the examined cows were subjected to metabolic stress, their organism compensated for this state in a relatively successful manner.
Tab. 1 presents the results of logistic regression analysis, where the influence of AcC on BWC and milk performance traits was determined. Generally, elevated AcC was related to higher body weight loss, slightly higher MY, higher FC, considerably lower PC and definitely higher FPR. Taking into account the parti-cular dates of examinations, the most divergent from 1.0 values of OR were noted for all dependent varia-bles in the case of AcC on the 60th day after calving.
Except for MY, OR values for other parameters were consequently slightly higher or lower than 1.0 on all examination dates. For MY in turn, OR was higher
than 1.0 for AcC on 5th and 30th day after calving, while
it was lower than 1.0 on the other two dates. Higher body weight loss in cows with elevated acetone levels during the initial lactation period should be explained by the fact that elevated levels of ketone bodies lower the appetite (13), which otherwise is already low at this time. The results obtained by other authors concerning the influence of elevated levels of ketone bodies on milk yield are also not consistent, since some of them claim that an increased level of ketone bodies decreases milk yield (13, 14, 21), others indicate a lack of rela-tionship (25), and others claim that this is a positive relationship (18, 33). The changing influence of ele-vated levels of ketone bodies in an organism in sub-sequent months of lactation on milk yields seems to be primarily results of changes in insulin levels in an organism, as well as lowered reactivity to this hormone. Among other things, in the phase of increasing daily milk yields during lactation which is observed in its first month, the cow organism does not react at all or reacts poorly to ketosis with milk production drop, since it is intensively stimulated by considerably lowered insulin levels observed naturally at this time (4, 16, 22). However, later the level of that hormone returns to normal, and thus the organism starts to react to an elevated level of ketone bodies with an increase in milk yield, while the critical moment occurs somewhere about midway through the second month of lactation (14, 21). Relationships between AcC and FC, PC and FPR presented in tab. 1 find confirmation in the studies of a number of other authors (9, 18, 19, 25). Positive relationships between ketone body levels and the fat content in milk in the initial lactation period is explained by the fact that a considerably increased amount of non-esterified fatty acids, as well as of beta-hydroxy-butyric acid which is formed in large amounts at this time as a result of the intense lipolysis of reserve fat, is aimed by an organism at milk fat synthesis (3, 22, 28). In turn, a lowering effect of ketosis on protein content in milk during early lactation is mainly a
re-Tab. 1. Results of logistic regression analysis (odd ratios) of blood acetone concentration on body weight changes and milk production traits s t n e m e r u s a e M e n o t e c a d o o l b f o ,) C c A ( n o it a rt n e c n o c m u tr a p t s o p s y a d s e g n a h c t h g i e w y d o B s y a d 0 6 -5 n e e w t e b ) C W B ( m u tr a p t s o p n o it a t c a l f o s y a d 0 0 1 t s ri f e h t n i s ti a rt n o it c u d o r p k li M ) Y M ( d l e i y k li M Fatcontent(FC) Proteincontent(PC) Fa/tproteinraito(FPR) £445vs.>445g/day £2650vs.>2650kg £4.00vs.>4.00% £3.05vs.>3.05% £1.32vs.>1.32 5 13..00 11..03 11..01 0.61(¯.01.6) 12..07 0 3 11..90 13..03 11..05 0.41(¯.02.6) 13..01 0 6 13..05 0.21(¯.05.7) 15..00 0.0131(.¯078.0) 211.80.9 0 9 11..04 0.71(¯.01.5) 11..40 0.31(¯.03.0) 618..03 l a t o T 13..60 11..80 13..04 0.0141(.¯071.4) 114.40.7
sult of insufficient intake of energy easily available for microbiological protein synthesis, which after digestion in further segments of alimentary track is transferred in the form of amino acids to the udder (22). A conducive factor in a decrease in milk protein synthesis is also the suppressive influence of ketone bodies on the mobilization of protein reserves in an organism (20, 28), and the fact that some amino acids are subjected to oxidation processes in considerable amounts during NEB and are therefore the precursors of ketone bodies (17, 22). Since milk fat and protein are formed via different metabolism pathways, it is claimed that FPR or another index concurrently invol-ving both components is a considerably better indicator of the adaptation of cows to metabolic stress during the initial period of lactation than the level of each of them separately (11, 18, 19, 25, 29).
Tab. 2 presents the results of logistic regression ana-lysis, where the reverse model in terms of the calcula-tions for which results were presented in tab. 1 was used. Namely, acetone levels in blood were assumed to be a variable dependent on body weight changes and milk performance traits. This analysis was perfor-med due to previously signaled inconsistent literature information (13, 14, 18, 21, 25, 33) concerning the background of the causal relationship between elevated concentration of ketone bodies in blood and milk yield. It may be concluded from the data obtained that BWC and MY did not influence AcC, since OR was always equal to 1.0 in the exposed group. Relationships demonstrated for milk composition parameters were, in turn, similar to those noted in tab. 1; however, the estimated OR values differed as a rule considerably less from 1.0. An exception are the data from the 30th
day after calving, when OR values for PC and auto-matically for FPR differed even 3-fold more from 1.0 than analogous values of this estimator in tab. 1. Thus, the results presented in tab. 1 and 2 prove that causal relationships between AcC and BWC were unilateral, while relationships between AcC and milk composi-tion parameters were mutual, whereby, except on the
30th day after calving, the acetone concentration in milk
was more a reason for milk composition variation than its result. The literature does not provide information that would allow an unequivocal interpretation of the biological mechanism of the consecutive character of the relationships between fat and protein concentra-tions in milk and ketone body levels in an organism during the discussed period of lactation. It may be sup-posed that this is associated with the observed reversi-bility of reactions related to ketone body metabolism (20, 28), the strategic aim of which is the prevention of an excessive loss of energy and protein reserves. It is symptomatic that depletion of labile protein reserves (6, 8) in cows of the Holstein-Friesian breed is usually observed during periods of lactation when an especially distinct influence of protein concentration in milk on AcC is noted which almost overlaps with the highest NEB (15). It was thus possible in this study that in order to protect further protein loss from an organism, homeorhetic mechanisms meant that at this time the priority was utilization of free amino acids (metabolic protein) for milk protein production, and also other anabolic processes excluding their oxidation and related ketogenesis.
Tab. 3 presents the results of analysis where fertility parameters were assumed to be dependent features. In total and on all blood sample collection dates, higher AcC caused an increase, and thus deterioration, in both indices (OR > 1.0). Definitely the largest relationships were noted for the examinations performed on the 90th
day after calving (OR equal 8.3 for CC and 6.4 for NSC), while on earlier dates OR values in an exposed group did not exceed 1.9. A negative influence of an elevated level of ketone bodies on the fertility of cows has been noted by numerous authors (1, 10, 12, 14, 25). It was simultaneously determined that this rela-tionship is higher in primiparous than in multiparous cows (10). Similar to the present study, it was also observed that the effectiveness of insemination is related to the highest degree with the cases of elevated levels of ketone bodies in the period of cow
insemina-Tab. 2. Results of logistic regression analysis (odds ratios) of body weight changes and milk production traits on blood acetone concentration , g n il p m a s d o o l B m u tr a p t s o p s y a d e n o t e c a d o o l B n o it a rt n e c n o c L /l o m m ,) C c A ( s e g n a h c t h g i e w y d o B t s o p s y a d 0 6 -5 n e e w t e b y a d / g ,) C W B ( m u tr a p n o it a t c a l f o s y a d 0 0 1 t s ri f e h t n i s ti a rt n o it c u d o r p k li M ,) Y M ( d l e i y k li M g k Fatcon%tent(FC,) Prot(ePinCc,)o%ntent Fa/tpr(oFtPeRin)raito 5 > 4£ 400.. 11..00 11..00 11..02 0.61(¯.01.6) 21..50 0 3 > 4£ 400.. 11..00 11..00 11..50 0.11(.¯008.0) 111..04 0 6 > 4£ 400.. 11..00 11..00 12..90 0.041(.¯0028.2) 110.50.9 0 9 > 4£ 400.. 11..00 11..00 11..06 11..10 21..40 l a t o T £ 4> 400.. 11..00 11..00 11..01 0.11(¯.07.4) 18..03
tion (12). The mechanism of the influence of ketosis and its related changes in an organism on fertility firstly involves lowering the level of glucose, IGF-1, insulin and leptin, which in turn cause disorders in GnRH/LH secretion, negatively influence the reacti-vity of ovaries to FSH and lower steroidogenesis (7). Also, the lowered reactivity of an organism to insulin has a direct influence on the reproductive functions of cows (24). Apart from that, fertility decrease is a result of an impairment of ammonia detoxication in the liver, which accompanies ketosis (31), and is also due to lowered resistance to infectious diseases caused by the damaging impact of ketone bodies and free radicals on the immunological system (32). It may be inferred from tab. 3 that changes in body weight and milk yield were not related to reproduction performance para-meters. The reason was surely a lack of influence of BWC and MY on acetone levels in blood (tab. 2). It was also noted in the studies of other authors that taking into account concurrently milk performance traits, changes in body weight/condition, the level of ketone bodies as well as other traits being an evidence of energy balance in early stage lactation, FPR or other indices including concurrently FC and PC have the largest relationship with cow fertility (11, 18, 26, 29). In conclusion, the present study demonstrates that among the analyzed parameters FPR is the best early indicator of metabolic stress observed in dairy cows in an early stage of lactation due to the occurrence of NEB at this time. Similar diagnostic values may be
attributed to AcC; however, possibilities of the appli-cation of this parameter are considerably lower in prac-tice. The option of FPR application is very attractive due to the fact that new generations of devices for re-gular milking enable every-day automatic measurement of fat and protein contents in milk, which creates the perspective of a considerable increase in the speed and accuracy of diagnosis.
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Tab. 3. Results of logistic regression analysis (odds ratios) of blood acetone concentration, body weight changes and milk production traits on reproduction parameters
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