Functional features of platelets at the beginning of ontogenesis in purebred calves of the Yaroslavl breed

. After birth, calves develop an increased ability of platelets to aggregate. At the same time, during the newborn phase, the level of inactive platelets in the blood of the Yaroslavl breed calves slightly decreases. The total intravascular activity of platelets in their blood during the study increased by 10.4%. The number of various platelet aggregates suspended in the blood of calves during the observation showed an upward trend. This was caused by an increase in their platelet thromboxane synthesis, an increase in the number of mobile adenosine phosphates in platelet granules and an increase in actin and myosin levels in them. As a result of the study, it becomes clear that for the Yaroslavl breed calves during the first 10 days of life, an increase in the severity of platelet aggregation and secretion is characteristic. Apparently, this helps to create optimal conditions for them to realize hemocirculation and metabolism, and therefore for the successful growth of animals of this breed.


Introduction
The normal functioning of the whole organism can only be in conditions of optimal hemocirculation [1][2].A serious role in it is played by the state of platelets.Their physiological characteristics largely determine the optimum microcirculation [3][4] in all productive animals [5][6].In earlier studies, it was noted that the functional capabilities of platelets can change throughout life, under aging conditions, as any dysfunctions form, with the development of signs of pathology, the appearance of vasopathy and during any medical procedures [4].The dynamics of the level of functional activity of platelets in cattle during ontogenesis remains not fully elucidated.There are only isolated studies on the evaluation of platelet parameters in different animals of different ages and, as a rule, without connection with existing genetic features [5].These unrelated data do not provide an opportunity to get a holistic view of the age-related features of platelet function in cattle and create the need for further observations.The special significance of such studies is associated with the great physiological importance of platelets in the regulation of blood movement in capillaries [6].At the same time, the implementation of microcirculation is very important for the realization of the growth and development of animals, the state of their internal organs and the dynamics of their productive indicators [7].Taking into account the striking morphofunctional differences between the breeds of farm animals and the undoubted importance of platelets to ensure hemostasis at all stages of growth and development, it was justified to trace the state of platelet indicators in young dairy cattle breeds at the very beginning of ontogenesis.
The aim is to establish the characteristics of platelet counts of Yaroslavl breed calves during the newborn phase.

Materials and methods
Research was conducted in strict accordance with ethical principles established by the European Convent on protection of the vertebrata used for experimental and other scientific purposes (adopted in Strasbourg March 18, 1986, and confirmed in Strasbourg June 15, 2006) and approved by the local ethic committee of All-Russian SII of Physiology, Biochemistry and Animals' feeding (Record №11, dated December 4, 2015).
The work was carried out on 34 calves belonging to the Yaroslavl breed, which were born from cows of normal morphofunctional status as a result of the second or third pregnancy.The young animals were examined with an assessment of hematological parameters during the study five times: at the age of one to two days, at the age of three to four days, at the age of five to six days, at the age of seven to eight days and at the age of nine to ten days.
The time of occurrence of platelet aggregation (AP) was determined using a visual micrometer in response to several inducers: ADP (0.5 x 10 -4 M), collagen (dilution 1:2 of the main suspension), adrenaline (5.0x10 -6 M), thrombin (0.125 u/ml) and ristomycin (0.8 mg/ml).Aggregation in blood plasma was determined after its standardization by platelet concentration to a value of 200x10 9 platelets/l.The level of platelet activity in the blood was assessed by performing standard phase contrast microscopy.According to the data obtained, the Student's criterion was calculated.

Results and discussion
The observed young animals had some tendency to increase initially small hemostatic platelet capabilities.The number of inactive platelets in the blood of animals during the study showed a downward trend.During the study, the volume of activated platelets in the blood of young animals increased by 10.5%.Platelet aggregates of any size in their blood increased slightly (Table 1).Note: p -is the reliability of changes in the parameters taken into account compared to the first study.
In the following tables, the designations are similar.
In the examined calves at the beginning of the observation, AP under the influence of collagen developed in 32.3±0.14 s, slightly accelerating in the subsequent and last study, reaching 30.5±0.14 s.A similar tendency to reduce the AP time was noted in response to ADP and ristomycin in the final study reaching 36.6±0.13 s and 46.6±0.15s,respectively.The platelet aggregation response to the action of thrombin and adrenaline also tended to accelerate to levels of 49.6±0.13s and 92.1±0.14s,respectively (Table 2).Some activation of AP in animals during the neonatal phase was found to be associated with increased generation of platelet thromboxane.This was indicated by the growth of AP in the conditions of a simple transfer test, which reached the level of 34.2 ± 0.09% on the 9th-10th day of life.This was possible in the observed calves due to a certain increase in the enzymatic capabilities of cyclooxygenase and thromboxansynthetase in the platelet composition.This was indicated by an increase to the level of 83.6±0.13% of the AP level in the conditions of a collagen-aspirin test, which allows indirectly establishing the biological properties of the cyclooxygenase enzyme in platelets.At the same time, the severity of AP recovery in the collagen-imidazole test, which allows determining the enzymatic capabilities in platelets of the enzyme thromboxansynthase, also increased in calves during follow-up to 44.6 ± 0.12% (Table 3).Initially, a small content of ATP and ADP in calves in platelets during the newborn phase had a tendency to grow, reaching the level of 5.67±0.019and 3.46±0.009mmol/10 9 platelets by the end of observation.The intensity of their secretory release from platelets during the newborn phase tended to increase to 34.9±0.14% and 45.6±0.20%.
The amount of actin protein and myosin protein at the very beginning of observation in inactive platelets of young animals turned out to be 31.4±0.10%and 14.5±0.13% of the total protein in the platelet.By the end of the follow-up in calves, these indicators increased and amounted to 33.5±0.10% and 16.5±0.15% of the total protein in the platelet.During newborn calves, a slight increase in the severity of the formation of actin and myosin in platelets under the conditions of the aggregation process was demonstrated.

Discussion
Previous studies have revealed the serious significance of hematological indicators for the viability of any biological objects.Particular importance among them has recently been attached to hemostatic mechanisms that play an important role in maintaining homeostasis in mammals [8].Platelets are recognized as a very important component of hemostasis, which, as a result of the formation of aggregates, can affect blood flow in vessels of different diameters and thereby regulate it.The functional capabilities of platelets in young animals of highly productive breeds of cattle of different breed affiliation have so far been poorly studied.This was the motivating reason for carrying out this study on the young of the Yaroslavl breed.
Registration of AP in young animals in response to collagen and ristomycin showed its optimal level with a tendency to accelerate the process.This was a consequence of the presence of an unexpressed ability of the platelets of these calves to adhere with its tendency to increase during the newborn.Apparently, this reinforcement was provided by the work of two mechanisms [9].The work of the first mechanism is indicated by the detected tendency to the early onset of aggregation in blood plates under the influence of collagen on them.This was possible as a result of a gradual increase in the number of molecules of glycoproteins Ia-IIa and VI, which are receptors for collagen, on the outer membranes of the platelets of calves during the newborn phase.Another mechanism for increasing the severity of platelet adhesion in newborn calves should be considered the growth of receptors (GPIb) on platelet surfaces that can interact with Willebrand factor molecules in combination with the growth of this compound in their blood.The presence of this mechanism was indicated by the reduction in the time of AP that occurs when exposed to ristomycin in the observed animals [10].
A certain tendency to an increase in platelet aggregation found in the examined young was largely the basis for the protection of his body from blood loss.Their slight acceleration of AP under the action of a strong stimulant (collagen or thrombin) is provided by an increase in the density of receptors on platelet structures while increasing the activity of phospholipase C and enzymes implementing the phosphoinositol pathway of platelet activation [11].Under these conditions, in the platelets of the Yaroslavl breed calves, there is apparently an increase in the synthesis of inositol triphosphate, which causes them to activate the development of self-assembly of actin and myosin and their sliding relative to each other [12].
Under the influence of weak stimulants (ADP and adrenaline) the examined animals have platelet aggregation, experiencing some tendency to earlier development.Apparently, it was based on a gradual increase in the number of receptors for inducers on their platelet membranes, while increasing the expression of fibrinogen receptors (GPIIb-IIIa) on platelets and increasing phospholipase A2 in them [13].The latter circumstance, combined with the found tendency to increase cyclooxygenase and thromboxane synthetase, contributed to a certain increase in the release of arachidonate from platelet membranes, which could be used for the synthesis of thromboxane A2.Activation of this synthesis in the Yaroslavl breed calves was accompanied by an increase in the functional properties of cyclooxygenase and thromboxansynthetase located in platelets.
A very important mechanism for the development of a tendency to increase the activity of AP during the newborn in young Yaroslavl breed is also a tendency to increase actin formation and myosin formation in their platelets under aggregation conditions.Also an important role in this was played by the emerging tendency to intensify the release of ATP and ADP from granules contained in the cytoplasm of platelets.
During the newborn period, calves showed a tendency to increase the number of active types of platelets in the blood.This happened as a result of activation of their receptors to different aggregation inducers.The found tendency to increase intravascular platelet activity additionally indicated a certain increase in the exposure of vascular subendothelial collagen and an increase in its contact with platelets.This was both the cause and consequence of the tendency for calves to grow freely moving aggregates in their blood [14].Some acceleration of platelet aggregation found in the observed animals should be considered as an adaptive process aimed at increasing platelets that have undergone activation of aggregates and have arisen on their basis, which are required to exclude the risk of bleeding in them.
The established level of hemostatic manifestations of platelets in the blood of calves belonging to the Yaroslavl breed during the newborn phase is consistent with the separate information available in the literature on adhesive and aggregation manifestations in cattle without specifying the breed.There is reason to assume that this dynamics is determined not only by the state of platelet aggregation mechanisms, but also by changes in vascular disaggregate effects on platelets.

Conclusion
At birth, low biological capabilities of platelets are characteristic of calves belonging to the Yaroslavl breed.Its level should be considered functionally sufficient for the normal implementation of the microcirculation process in them and maintaining an optimum flow of nutrients and oxygen to the tissues.This situation in calves is associated with a low intensity of the mechanisms that implement the hemostatic properties of platelets.Their small activity in the bloodstream throughout the entire newborn phase provided unexpressed hemostasis activity and optimum trophism of all internal organs, creating the necessary conditions for the development of young Yaroslavl breed.

Table 1 .
Platelet activity in the bloodstream of Yaroslavl

Table 2 .
Platelet aggregation in Yaroslavl calves during newborn

Table 3 .
Hemostatic mechanisms of platelets in Yaroslavl calves during newborn