Selection for milk yield increases the demand for energy and intensifies energy metabolism in dairy cows. One important factor in this case is the changes in the negative energy balance (NEB) and content of nutrients in the milk. In this regard, concentrations of NEFAs esterified to triglycerides and β–hydroxybutyrate (BHBA), as well as the release of C18:1 from the adipocytes, remain the basic parameters of the metabolic profile of dairy cows. Their content is linked to maintenance of energy homeostasis, which is associated with the synthesis of FAs in the rumen and mammary gland [
22]. In the early lactation stage, increasing production is conducive to the appearance of NEB. Adipocytes then release FAs that are usually unfavorable for the nutritional quality of milk fat. Ducháček et al. [
14] showed that NEB increased the share of hypercholesterolemic FAs in the milk of HF cows by 1.86 p.p. on average. Their study showed that NEB mainly leads to an increase in MUFA, in which case the difference was 1.81 p.p. In the case of UFA the difference was smaller, at 0.33 p.p. A similar tendency was observed in our study, but the milk of HF cows had higher content of MUFA and lower content of PUFA than in the study cited. The milk of the HF breed also had the highest content of C16:0 and C18:1, which is the main deposit in the adipocytes. This may also suggest that diet did not directly influence the FA profile in the milk. Additionally, Sobótka et al. [
23] showed a significant influence of lactation phase and breed on the content of FAs. However, significant correlations were found only in the case of SFAs and PUFAs. This did not fully coincide with our results. Sobótka et al. [
23] the lowest content of C16:0 was found in the milk of HF cattle. More C16:0 has been found in the Jersey breed and its HF hybrids. However, there was no effect associated with lactation stage. However, it is interesting that the C16:1 released from adipocytes appeared in greater amounts in the later stages of lactation. However, this can be explained by the greater availability of energy for C16:0 synthesis in the mammary gland de novo [
16]. Incomplete compliance of our results with the studies by Sobótka et al. [
23] may be explained by the research of Poulsen et al. [
24]. These researchers explained that environmental variation affects individual FAs of milk differently, based on both breed and feed quality. The dietary effect on FA composition is obvious, but as they suggest, an important factor is also the source of origin of the feed ingredients. Furthermore, Samkova et al. [
5] suggested that the shaping of milk FAs may be affected to a large degree by cow individuality, but also by the stage of lactation. According these studies, breed is a factor of lower significance. Samkowa et al. [
5] suggest that biochemical changes, especially biohydrogenation in the rumen, should be studied in greater detail. In light of these studies [
5,
23,
24], the obtained FA profiles of milk in our experiment could have been shaped by other environmental factors, as well as the production potential of the studied breeds and their reaction to the formation of NEB. During early lactation it stimulates the content of non-esterified FAs (NEFAs) generated from lipid tissue increases. It is usually proportional to the size of the energy deficit [
25,
26]. This leads to an increase in long-chain FAs in the milk, which enter the mammary gland with the blood. Craninx et al. [
2] reported a lower content of C16:0 in the milk of Holstein cows in a grazing trial. However, the diet used by the authors, containing hay and silage from grass and maize, did not affect the content of C18:1. This may suggest that the diet does not directly affect the share of C16:0, which is influenced by NEB. Craninx et al. [
2] showed that in early lactation, the milk of cows with higher yields and with higher fat content had a lower share of FAs, with less than 15 carbon atoms. In our study, this was confirmed by the positive correlations between the level of long-chain FAs (LCFAs) and C16:0 and milk yield (0.593, 0.632;
p ≤ 0.05) and NEFA content (0.689 and 0.682;
p ≤ 0.05). A study by Vanbergue et al. [
26] showed that spontaneous lipolysis was mainly dependent on the breed of cows and season of production. The study did not confirm the effect of the feeding intensity or its interaction with breed on the course of lipolysis. Although the authors found that the influence of breed on SFA and MUFA was greater, feeding intensity was also found to have a minor effect. This effect, however, varied depending on the production season. In cows with high postpartum milk production, we noted higher concentrations of BHBA and NEFAs in the blood. At the same time, the blood glucose levels were usually lower.
Lipomobilization in high-producing dairy cows up to the peak of lactation could reach a pathological range, disturbing the liver’s morphological and functional efficiency. Diokovic et al. [
12] demonstrated that in the early-lactation cows, there is a rapid increase of fatty acids in the liver. The authors observed growth the lipomobilisation markers, especially the serum β–hydroxybutyrate and free fatty acid concentrations. According to the authors, the liver steatosis was affected, which disturbed the synthesis of hepatocyte. Consequently, it led to weaker concentrations of glucose and an increase in triglycerides. This effect has induced some cellular lesions, as evidenced by significant increases in the serum albumin and bilirubin concentrations. Šamanc et al. [
27] showed that an NEB is conducive to excess accumulation of fat in the hepatocytes. This situation may be a major cause of endocrine disorders, including secretion leptin. This may result in impaired gluconeogenesis and reduced glycaemia. In comparison with our results, Vargová et al. [
28], who studied the hormone profile of Slovak Pied Cattle, reported higher blood glucose concentrations. However, up to nine weeks postpartum they observed a downward trend in NEFAs and BHBA, accompanied by a decrease in body condition score (BCS). The authors [
28] noted a positive correlation between BCS and leptin (0.360;
p ≤ 0.001). Contrary to the studies by Vargová et al. [
28], in our study we showed a negative correlation between BCS and leptin (−0.480;
p ≤ 0.05) and a positive correlation between daily milk production and leptin (0.417;
p ≤ 0.05). As demonstrated in our studies, the NEFA × leptin correlation (0.416;
p ≤ 0.05) indicated that a higher level of leptin led to a higher concentration of NEFAs released during the energy deficit. This may be related to decreased appetite. In consequence, reduced feed intake may result in a lower glucose concentration in the blood. This is explained by the negative leptin × glucose (−0.259;
p ≤ 0.05) and NEFA × glucose (−0.386;
p ≤ 0.05) correlations. This may indicate that the level of the appetite regulator leptin is linked to the production potential of the breed. In our study, a lower leptin concentration was usually noted just after calving, but it was higher in the groups reaching the peak of lactation with a higher yield. The obtained values of the correlation coefficient with leptin were relatively low, but they may suggest that the stronger appetite in individuals with lower leptin levels may prolong feed intake. In effect, this may be linked to better energy availability and to energy conversions that are beneficial for FAs. This indicates more rapid induction of lipolysis due to reduced appetite in cows with higher yields [
27,
28]. Reduced appetite and higher production potential may also explain the lower glucose level in the blood of animals during the peak of lactation and reaching this peak with a higher yield. According to authors such as Lock and Garnsworthy [
16], a greater energy deficit associated with milk production may reduce direct absorption of cis–9–C18:1 in the small intestine and limit processes involving desaturase in the mammary gland. Liefers et al. [
29] reported a lower leptin content in the blood of cows that produced more milk, as in our study. They also showed lower DMI in cows with a negative energy balance. Our results were not confirmed in this case, as the content of C18:1 as an indicator of NEB was usually lower in cows with lower leptin levels. This relationship is confirmed by the correlation of C18:1 × leptin (–0.428;
p ≤ 0.05). Buttchereit et al. [
30] also showed that strong mobilization of energy from lipid tissue is not only conducive to more intensive production of long-chain FAs, but can also reduce the protein content in milk. This is consistent with the tendency observed by Pupel et al. [
31] and with our results regarding levels of κ–casein. Our results for the content of the main FA fractions are in agreement with those reported by Petit and Côrtes [
32]. They found the highest BHBA level (on average 748 µmol/L) in milk with a higher content of fat and SFAs. The exception was a group whose diet included ground flaxseed. The milk of this group had the highest content of BHBA (1512 µmol/L) and high levels of MUFAs and PUFAs. In this case, the effect was explained by the greater availability of energy reaching the mammary gland, in which a considerable portion of these fractions is generated de novo. In this group of cows, Petit and Côrtes [
32] also showed the highest content of non-esterified FAs in the blood (337 µmol/L), although the production level in this case was one of the lowest (29.5 kg/d). The cows in this group, however, had the lowest glucose level in the blood, which in our study was negatively correlated with NEFA (−0.386,
p ≤ 0.05). Puppel et al. [
31] showed very strong negative correlations between the BHBA level in the blood and the content of CLA–9 and –10. Our results are consistent with those reported by Vanbergue et al. [
26], who studied the milk of Holstein and Normande cows up to the peak of lactation. The share of SFAs ranged from 61.8% to 73.5% and that of MUFAs from 23.0% to 34.7%. Vanbergue et al. [
26] also noted more intensive lipolysis of fat reserves and 1.1% to 5.6% higher content of cis–9 in the milk of cows fed less intensively. Adamska et al. [
4], in comparison to our study, reported a higher content of SCFAs (15.99 g/100g FAs) and a similar content of PUFAs (2.53 g/100g FAs) in the milk of Simmental and HF cows. However, the milk of the Simmental breed usually had a more beneficial composition, and also contained more branched-chain FAs. The milk of HF cows, on the other hand, had the most LCFAs (52.11 g/100g FAs) and MUFAs (26.32 g/100g FAs). The results of our study also correspond with those reported by Król et al. [
33], in which the milk of Simmental cows contained on average 30.53% SFA, which was on average 4.1 p.p. less than in the milk of HF cows. In the milk of Simmental cows, Król et al. [
33] showed higher levels of MUFAs and PUFAs, at 27.13%and 3.43%, respectively. Such large differences were not observed in our study, which can be explained by the use of a similar diet (TMR) in all breed groups.