Introduction

Animal health and herd productivity are the most difficult challenges that dairy producers confronting on a regular basis. The period around calving is critical due to the reduction in dry matter intake (DMI), increases in the demand of nutrients, energy and calcium (Ca²⁺) for the maintenance and synthesis of milk. Due to the reduction in DMI, the requirements of the animals cannot be met, and the deficit allows the animal to fall into negative energy balance (NEB).

At early lactation, the concentration of glucose in blood serum is low, with a parallel increase in the concentration of non-esterified fatty acids (NEFA), and ketone bodies¹. The most prominent circulating ketone body in ruminants is β-hydroxybutyrate (BHBA), which is used as an energy source in body tissues such as brain, heart², kidney and skeletal muscle³. However, the increase of BHBA above 1.2 mmol L^-1 is an indicator of subclinical ketosis in dairy cows⁴. The increase in plasma BHBA reduces circulating glucose in blood⁵ and increases the risk of ketosis, hypocalcemia, abomasal displacement and metritis with the consequent reduction in MY⁶.

Measurements of BHBA, Ca²⁺, and NEFA concentrations around calving may be potential indicators of the cow's ability to overcome metabolic challenges in the transition period, and possibly allow predicting some disease risks and possible MY losses at the start of lactation. Calcium concentrations demonstrate the ability of the cow to replace extracellular Ca²⁺ loss as a result of the milk production process, and the balance between bone, and the efficiency of absorption of insulin and Ca²⁺⁷. Non-esterified fatty acids serve as an indicator of mobilization of body fat and reflect the particularity of the cow to adapt to the NEB.

At the level of cow, reductions in serum Ca²⁺ concentrations, increases in NEFA and BHBA have been associated with an increased risk to contracting diseases⁸ and milk loss⁹. The cow-level thresholds of these metabolites have been used to identify individuals at risk of damaging their health and productivity. However, individual interventions to minimize the undesirable effects of NEB on hypocalcemia, ketosis or other metabolic disorders around calving are difficult to achieve. Based on this premise, the objective of this study was to determine the serum concentrations of β-hydroxybutyrate, Ca²⁺, and, non-esterified fatty acids, seven days before parturition, and the relationships between them and milk losses at seven and 14 d of lactation in Holstein-Friesian cows in confinement.

Material and methods

Results

Table 2 shows the thresholds and the number of the cows for BCS, lactation and MY of animals used in the study; whereas, Table 3 shows the number of repetitions, the mean and the standard deviations values of the blood serum metabolites concentrations of the animals considered in the study.

Table 4 shows the sampling dates, and the proportion of cows that were above and below the thresholds. The proportion of cows above the thresholds for BHBA at d-7 was less (P≤0.05) than those observed at d 7 and 14. In contrast, it was not observed differences between d 7 and 14.The proportion of cows above the thresholds for Ca²⁺ was greater (P≤0.001) for -7 and 7 d in comparison with 14 d. However, for NEFA, the proportion was greater (P≤0.001) in d 14 than the other days (224 vs 108 and 30, for 7 and - 7, respectively).

The proportion of cows below the thresholds for BHBA at d 7 and 14 was less (P≤ 0.05) than those observed at d-7. In contrast, it was not observed differences between d 7 and 14 (Table 4). The proportion of cows below the thresholds for Ca²⁺ was greater (P≤ 0.04) than - 7 and 7 d in comparison with 14 d. However, for NEFA, the proportion was greater (P≤0.001) in d-7 than the other days (306 vs 228 and 122, for 7 and 14, respectively).

Table 5 shows the metabolite concentrations observed in cows that were above and below the thresholds obtained for BHBA, Ca²⁺ and NEFA, on d -7, 7 and 14. In cows above the thresholds, the concentrations of BHBA at d 7 and 14 were greater (P≤0.02) than those observed at d-7. On the contrary, the concentrations of Ca²⁺ and NEFA at d - 7, 7, and 14 prepartum were not different (P≥0.05).

With respect to the cows below the thresholds, the concentrations of BHBA at d 7 and 14 were different (P≤0.02) compared with -7 d; on the contrary, no difference (P≥0.02) were observed between d 7 and 14. While for the same date, the Ca²⁺ concentrations were not different (P≥0.12) compared to the other two dates. The NEFA concentrations of d-7 were different (P≤0.01) to the concentration on d 7 and 14 postpartum. On the contrary, no difference was observed between d 7 and 14.

Cows on day seven after parturition showed high-risk with blood concentrations of NEFA. The relationship of blood concentrations determined on d-7 before calving and metabolic dysfunctions incidence in the first 7 d of lactation had a significant effect for the incidence of lameness (P≤0.01), with an OR estimate of 7.6 [1.50 - 38.74; CI = 95%; P=0.01] times more likely to present this clinical disorder when NEFA ≥0.5 mmol L^-1. It was also observed that 11 % of the cows with high concentrations of BHBH were lost approximately 0.37 kg cow^-1 d^-1 of milk [Odd ratio = 0.37; (0.14 to 1.01; CI = 95%; P=0.05)].

Discussion

The ketone bodies, with the BHBA as the main one, are known to be involved in the expression of ketosis, as depressants of feed intake and that affect negatively the fertility of dairy cows¹⁷. It was determined the serum concentrations of BHBA, Ca²⁺, and NEFA 7 d before parturition, and 7 and 14 after parturition, in order to establish the relationships between those concentrations and milk losses in Holstein-Friesian cows in confinement.

In the current study, the low proportion of cows above the BHBA thresholds on d - 7 in comparison with d 7 and14 of lactation was explained by the metabolic and endocrine adaptations of the cow during the dry period in the absence of the requirement of nutrients for MY; while in lactation; the metabolism of nutrients is related to negative energy balance (NEB) caused by MY and inadequate feed intake.

The necessary adaptation is reflected by changes in several blood parameters. As noted in other studies, the concentration of glucose decreases, whereas the concentrations of BHBA and NEFA increase, concomitantly with related changes in the endocrine system mainly insulin and glucagon¹⁸.

The difference in BHBA concentrations before and after parturition was also observed in other studies. Chapinal et al⁹ in a large multiregional study conducted in 55 stables, to validate the relationships between concentrations of BHBA with diseases at the beginning of lactation, observed that cows sampled -7 d were four times below the risk threshold for BHBA compared to those sampled seven days after parturition. The difference was explained by the metabolism of energy in different physiological states.

In the Chapinal et al⁹ study BHBA concentrations was similar to those observed here. In the present study, the serum concentrations of BHBA found were on average 0.73 with a variation of 0.20 to 3.84 mmol L^-1. The highest concentrations were obtained on d 7 and 14 of lactation in comparison with those obtained on d -7. The cow-level threshold of <0.8 mmol L^-1 used in the present study is similar to those suggested by Chapinal et al⁹ and Ospina et al¹⁹. Conceivably, subclinical ketosis may start at levels of BHBA greater than 1.0 mmol L^-1; however, the decision to set an appropriate subclinical threshold using serum or plasma BHBA appears to be somewhat arbitrary.

Kelly²⁰ suggested that 1.0 mmol L^-1 be used to separate cows with low and high BHBA concentrations; whereas, Suthar et al²¹ selected a subclinical ketosis threshold of 1.2 mmol L^-1 of BHBA. Because increased ketone bodies post calving is considered to be part of a normal metabolic response to increased energy demand, it seems that a cut point to define high ketone body concentrations should be based on both production and health impairment.

Other factors such as age of the cow are also part of the explanation of the high levels of BHBA in early lactation. In the current study, cows with three or more (2.5 vs 3.6 ± 1.5) lactations were more productive than young cows as a consequence a greater proportion were above the risk threshold (approximately 19 %). Older cows (> 4 lactations) consume more feed than younger cows, mobilize more glucose and respond with greater increases in BHBA due to the needs of lactation¹³.

According to these criteria, in the present study approximately 3, 7 and 8 % cows would be considered with subclinical ketosis; because their BHBA concentrations were higher than 1.0 mmol L^-1 for d -7, 7 and 14, respectively. It can be a call for attention to health and the possible requests for milk in the herd under study. Particularly, the herd in study has the most productive and reproductive records of the area, which means that other herds with lower parameters could present subclinical ketosis in greater proportions in the study area. Calcium plays a fundamental role in MY, because it is involved in the transmission of nerve impulses, contraction of muscles, blood coagulation, secretory activity of cells, cell differentiation, immune response, and enzymatic activation²².

In the current study, the proportion of cows above the Ca²⁺ thresholds were lower for d 14 compared to 7 and -7; this may be due to the mobility of Ca²⁺ during the end of gestation only has very slight fluctuations because the changes in the requirements for the mineral are not very variable. On the contrary, in the days after parturition, the needs for the metabolite increase dramatically as the synthesis of the milk produced increases. According to the NRC¹¹ at the start of lactation, approximately all cows are on a negative Ca²⁺ balance, so as lactation progresses and MY increases in volume, as a consequence the need for mineral increases.

Van't Klooster²³ indicated that the absorption of Ca²⁺ increased 22 % towards the end of pregnancy to 36 % by d 8 of lactation in cows that consumed total mixed ration (TMR). What represented approximately 1.6 times increase in Ca²⁺ absorption. After that period, the changes were small and in response to increases in MY.

Chapinal et al⁹ indicated that Ca²⁺ effectively tends to increase in order to cover the demand of the mineral for milk synthesis. This study included 48 herds, of which 33 % were above the risk threshold when Ca²⁺ was determined on d -7, compared to 40 % of them that were above the same threshold when determined Ca²⁺ on d 7 postpartum.

In the present study, serum Ca²⁺ concentrations were approximately 2.46 mmol L^-1, with a wide range of variation from 1.24 to 4.98 mmol L^-1. As indicated, previously, the Ca²⁺ threshold was <2.3 mmol L^-1, suggesting that a high proportion of cows were at risk of presenting subclinical hypocalcemia (serum Ca²⁺ concentration <1.8 mmol L^-124; although the signs of milk fever were imperceptible.

As indicated above, the herd under study has the highest productive indicators of the dairy basin, and still presented a high number of cows prone to subclinical hypocalcemia. The inference could be true for herds with similar management conditions, where, possibly that condition can be a problem that limits MY. Another part of the explanation is also related to the age of the cow. Adult cows with more than four lactations (approximately 19 %) were more prone to hypocalcemia and were above the proposed threshold for Ca²⁺. In support of the above, Venjakob et al²⁵ observed that primiparous cows with serum concentrations of Ca²⁺ < 2.0 mmol L^-1 had no effect on MY, while adult cows not only showed hypocalcemia, but also produced less milk (- 2.19 kg animal^-1 d^-1) than cows free of the disease.

Neves et al²⁶ observed that prepartum cows with concentrations of Ca²⁺ ≤ 2.4 mmol L^-1, had higher risks of being classified as subclinical hypocalcemia one week before parturition. The authors indicated that the Ca²⁺ threshold (≤ 2.4 mmol L^-1) is required for the identification of animals in prepartum with a higher probability of presenting subclinical hypocalcemia. Herd-level studies using this cut-off point could establish objectives to measure the success of preventive strategies.

Additional studies are needed to determine the sampling times of blood around parturition, and to evaluate the relationship between fatty acids and Ca²⁺ in the prepartum. In modern dairy farms, the periparturient period (approximately 3 wk before and after parturition) presents the highest risks for developing diseases and mortality in cows. Ferguson²⁷ suggested that approximately one of two cows suffer adverse effects on their health, with approximately 75 % of the diseases occurring during the peripartal period¹³. The most common metabolic diseases, such as hypocalcemia and hyperketonemia, have been shown to alter the cow's immune system, increase risks for other diseases, and reduce productive behavior¹⁷.

In the current study, approximately 30 cows were above that threshold for NEFA on d -7, while for days 7 and 14 of lactation, number of cows increased from 108 to 224, respectively. The postpartum increase in NEFA levels could be explained by the increase in nutritional requirements due to the particular needs of lactation. This effect is related to the fact that most cows are in NEB, and they need to mobilize their body reserves of lipids in order to cover their energy needs^11,28.

Chapinal et al⁹ from a larger study in cows and locations than the present study, but based on same BHBA, Ca²⁺, and NEFA thresholds concluded that approximately 11 % of the herds studied were above the risk threshold on d -7 compared with 23 % of herds on day seven of lactation. The authors concluded that 5 to 50 % of the animals sampled with NEFA levels above the thresholds both a week before and after were associated with risks of abomasal displacement, milk loss and pregnancy reduction at the first insemination.

In the present study, NEFA levels averaged 0.59 mmol L^-1 with a range between 0.12 and 2.95 mmol L^-1. This suggests that the cows in the study would be at risk of losing milk at the beginning of lactation, mainly due to some association with hypocalcemia and hyperketonemia due to the circulating levels of NEFA.

The results obtained in the current study do agree with those from other studies. Ospina et al¹⁹ found that NEFA concentrations > 0.3 mEq L^-1 from d 14 to 2 prepartum, and NEFA > 0.6, and BHBA > 10 mg dL^-1 from 3 to 14 d postpartum, both prepartum and postpartum values above these thresholds were associated with increases in ketosis, metritis, abomasal displacement, and placental retention.

In the current study, it was search through conditional logistic regression to relate the concentrations of metabolites with metabolic dysfunctions around parturition. However, the associations obtained were not significantly important and due to this they were not reported. One significant unique observation was as the blood NEFA increased (≥ 0.5 mmol L^-1) there was an increase of up to 7.6 [Odd ratio = 7.61; (1.50 to 38.74; CI = 95%; P=0.01)] times the chances of laminitis occurrence; this may be due to the fact that lameness produces fever and pain in the hooves, which alter resting and feeding behavior of the animals^29,30.

Fever reduces the cow's appetite and pain in the hooves impair the ability to walk and look for feed. The deficiency of food increases the possibility of the removal of body reserves, increasing the free fatty acids.

Results from the current study were observed by others. Collard et al²⁹ indicated that plasma samples with NEFA concentrations of 0.6 to 0.8 mmol L^-1 were associated with lameness presence around parturition in Holstein-Friesian dairy cows fed TMR.

The results obtained in the present study in relation with the levels of NEFA and laminitis are contradictory with other reports in literature. Calderon and Cook³⁰ reported no relationship between lameness and NEFA in Holstein-Friesian cows around parturition in a mattress-bedded commercial free stall facility. They also observed that lame cows had longer lying times throughout the transition period and notably for 3 d before and after parturition. Lameness was also associated with a greater risk for ketosis in the study farm, as evidenced by the elevated BHBA concentration found in cows.

The results obtained in the present study offer the opportunity to examine the effects of pre and postpartum concentrations of NEFA and BHBA on MY at the cow level. The identification of these prepartum levels will allow the dairy producers to improve their strategies of nutritional management, in order to avoid losses in MY or to avoid the presence of metabolic disorders such as laminitis. The results obtained also show how the metabolism state influence health cow during the transition period suggesting that nutritional management has to be carefully reviewed due to its impact on subsequent lactation.

Conclusions and implications

High proportion of cows are above the thresholds of β-hydroxybutyrate and non- esterified fatty acids, and most of them were also deficient in calcium, when determined one week before parturition. High β-hydroxybutyrate concentrations could promote losses in MY up to 0.37 kg cow^-1d^-1 seven days after calving in Holstein-Friesian cows. High non-esterified fatty levels were associated with 7.6 times more risk of laminitis. The risk thresholds for each metabolite were not associated with the amount of milk lost at d 14 after calving in Holstein-Friesian cows.