Impact of Craniocerebral Hypothermia and Cryopreserved Cord Blood on Reproductive Function of Male Rats with Chronic Alcohol Intoxication

In this work, we have studied the impact of chronic alcohol intoxication (CAI) on reproductive function in male rats. Prolonged alcohol abuse has been shown to signifi cantly reduce the manifestation rate of sexual activation and testosterone level in blood serum of male rats with CAI following exposure to a receptive female, as well as to change their seminal fl uid composition. The rhythmic craniocerebral hypothermia (rCCH), combined with the administration of cryopreserved cord blood leukoconcentrate (cCBL) ensured to a greater extent the dynamics of functional integrity recovery of the CAI-altered reproductive system in male rats, if compared to each of these methods used solely. The authors have hypothesized the rCCH and cCBL to activate the hypothalamicpituitary system, aff ecting thereby the testosterone synthesis and spermatogenesis stimulation.

the impact on not only metabolic processes, but functional state of integrated systems (nervous, endocrine, immune, reproductive) as well. One of the major consequences, associated with alcohol abuse is sexual dysfunction. Men of all ages experience sexual dysfunction as a serious psychological trauma [7].
According to the reported data, the frequency of sexual dysfunction in people suff ering from alcoholism is up to 83.0% [10,14,22]. Chronic alcohol intoxication (CAI) can be the main cause of male infertility associated with alcohol abuse in most cases (96%). This is due to a functional state of endocrine glands (alcohol interferes with testosterone (Ts) release from Leydig cells) [6] and the neuroendocrine regulation impairment of the early stages of spermatogenesis, a decrease in total number of mature spermatozoa and increasing pathospermia and asthenozoospermia [15].
The CAI was established to aff ect the reproductive system both directly (via a toxic eff ect) and indirectly (by modulating a functional state of the hypothalamic-pituitary-gonadal system) [9,18], to cause a prolonged neuroadaptation, and alter the neurotransmission and synaptic plasticity [1]. Therefore, restoring the functional integrity of reproductive system necessitates an active infl uence on neuromediator systems in order to ensure an adequate activity of neuroendocrine functions. The therapy of hypothalamic neuroendocrine syndrome should include the non-standard methods, enabling to control targetedly the complex of central nervous system (CNS) structures, which provide the integrity of functional responses of the pineal gland-hypothalamus-pituitary system. We suggested the rhythmic craniocerebral hypothermia (rCCH) as likely referred to these methods [11,12].
The fi ndings, obtained at the Institute for Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of Ukraine (IPCC of NAS of Ukraine) have demonstrated the rhythmic cold exposures to enable obtaining a ther-moregulatory response, expressed as an adequate reaction of neurohumoral processes of hypothalamic-pituitary system. This thermoregulatory response ensured a change in the direction of neurotransmitter processes, neurohumoral and autonomic responses in dynamics of exposure [11,12].
The studies implemented at the IPCC of NAS of Ukraine have also shown an effi cient application of cryopreserved cord blood (cCB) to correct various disorders of body's functional systems, especially neuroendocrine and reproductive ones [2,4,13].
Готову кріоконсервовану суспензію лейкоцитарних клітин (лейкоконцентрат) кордової крові людини [12] отримували в кріобанку ІПКіК НАН України. enhance the manifestation of central (paradoxical) eff ects of systemically administered drugs and to potentiate their action, as well as the feasibility to apply rCCH and cCBL in therapy of reproductive disorders, induced by toxic CAI eff ect [11,12]. Therefore, we aimed here to determine the infl uence of rCCH, cryopreserved cord blood leukoconcentrate (cCBL) and their combined use on the CAI-injured reproductive function in male rats.

Materials and methods
The study was performed in adult 12-monthold male rats. The experiments were conducted at the scientifi c departments of the IPCC of NAS of Ukraine in accordance with the Law of Ukraine 'On the Protection of Animals Against Cruelty' (№ 3447-IV of February 21, 2006) in compliance with the requirements of the Bioethics Committee of the IPCC of NAS of Ukraine, consistent with the provisions of the'European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientifi c Purposes' (Strasbourg, 1986).
The intact animals were housed in the animal facility with a standard diet ad libitum.
The rats were chronically intoxicated with alcohol starting from 3-month age [5] and during the entire experimental period.
The animals were divided into 5 groups (n = 15 in each): the group 1 comprised the intact rats; the group 2 was the rats with CAI (CAI control); the group 3 consisted of the rats with CAI, received the rCCH procedure (rCCH); the rats with CAI, with administered cCBL (cCBL) were in the group 4; the rats with CAI, who underwent the combined methods of exposure (rCCH + cCBL) were included in the group 5.
The rCCH were done with the Fluidokranioterm ATG-01 device (Russia), which ensured an intermittent supply of cold air (4-6°C) at an exposure frequency of 0.05-0.2 Hz. The rhythmic CCH was conducted once, and a rectal temperature did not fall below 35°C [2].
The cryopreserved suspension of leukocyte cells (leukoconcentrate) derived from human cord blood [26] was provided by the Cryobank of the IPCC of NAS of Ukraine (Ukraine).
The post-thaw cCBL samples were administered once intraperitoneally at a dose of (3-5) × 10 7 cells / kg of body weight.
The rCCH and cCBL were administered together, observing the following order: the rCCH procedure was performed fi rst, then a day later the animals were injected with the cCBL.
The rats were sacrifi ced to day 30 after the last procedure.
Еякулят для досліджень отримували у щурів методом окситоцинової стимуляції (внутрішньочеревно в кількості 0,2 мл/тварину) [6]. Краплю еякуляту (≈50 мкл) збирали за допомогою очної піпетки або шприца без голки об'ємом 1 мл. Отриманий еякулят розводили фізіологічним розчи-Sexual motivational behavior of male rats was studied in the evening at low lights in the standard plastic cage (52 × 33 × 20 cm), divided into 2 compartments by a transparent perforated barrier, through which the olfactory and visual contacts between animals could occur. The time, during which the male actively examined the barrier and a number of its approaches to it, were recorded [3,25]. A 10-min testing was performed when no female was present behind the barrier (assessment of a spontaneous activity) and while a receptive female was in the adjacent compartment (evaluation of a female-stimulated male activity near the barrier).
Under conditions, when any contact between animals was excluded (receptive female was separated from male by a transparent perforated barrier), we assessed the pronouncement of sexual motivation by male behavior within 10 min, as well as by hormonal activation of testosterone (Ts) level in blood serum, which increased in rats on the 20 th -40 th min following exposure to a receptive female [3].
To evaluate a hormonal component of sexual behavior (Ts level), the blood was sampled from tail, by cutting off 3-4 mm of its tip under light (for a minute) ether anesthesia. Blood was sampled under sexual activation (20 min after exposure to a receptive female at an adjacent compartment) and without it (baseline hormone level). The Ts content in blood serum was determined with the enzymelinked immunosorbent assay (ELISA) using the Testosterone-IFA standard commercial kits (XEMA Co. Ltd., Russia) according to the manufacturer's techniques.
For spermatogenesis assessment, there were determined the spermatozoa parameters (their total number in ejaculate, percentage of living motile, dead and defective spermatozoa). The ejaculate was examined with light microscope in Goryaev's chamber, the results were expressed as a percentage.
The ejaculate for study was collected from rats by oxytocin stimulation (intraperitoneally, 0.2 ml / animal) [8]. A drop of ejaculate (≈50 μl) was taken using a 1 ml eye pipette or syringe without needle. The resulting ejaculate was diluted with saline of 37°C up to 2 ml volume, then placed in a thermostat at 37°C for 30 min. Afterwards, 0.1 ml of diluted ejaculate was supplemented to the vial, containing 0.9 ml of saline [8,23].

Results and discussion
Sexual behavior is among the biologically relevant behaviors, realized as a certain sequence of behavioral acts. Male rats in the preparatory (search) phase, which allowed the animal to achieve the contact with the target object, demonstrated the following behaviors: the male moved along the barrier, sniff ed the female, got up on its hind legs, touched the barrier with one or both forelegs, poked its nose into the holes or gnawed their edges in an attempt to get over the barrier and fi nd its way to the female. This behavior is the basic behavioral feature of sexual motivation, and a number of approaches to the barrier mostly refl ects the motor activity and general arousal of animal [3,25]. Notably that a sexual motivation is inherent in men as well [20].
The results of component analysis of sexual motivational behavior in male rats with CAI demonstrated the indices of courtship behavior (a number of approaches to barrier and their duration) to signifi cantly diff er from those in intact animals with constant spontaneous activity (72 and 56.25%, respectively) ( Table 1). Thus, a long-term alcohol consumption signifi cantly (p < 0.05) reduced the interest to the opposite-sex species, by causing the so-called 'sex neutralization' eff ect.
The disorders in sexual behavior in CAI rats were likely the result of an altered metabolism of sex hormones, which formed the background for proper functioning of regulation centers of sexual behavior [20]. A reduced concentration of the primary male sex hormone (Ts) in blood serum entailed a decrease in sexual activity [23]. Table 1 showed the baseline Ts level we recorded in males of group 2 (CAI) to be 46.16% (p < 0.05) lower vs. the intact control, while after exposure to a receptive female, the stimulated Ts rate was 73.81% lower as compared with this index in group 1.
The performance of rCCH session signifi cantly increased the manifestation rate of sexual motivation in experimental males if compared with the group 2 (CAI). For example, the time of active examination of barrier by male and a number of its approaches to it in group 3 (+rCCH) exceeded the control indices of group 2 by 98 and 57%, respectively (Table 1). Herewith, the indices of baseline and stimulated Ts in males diff ered from similar indices in CAI rats by 28.57 and 72.72%, respectively.
The most pronounced alterations of sexual activity in CAI rats were observed after administering both rCCH and cCBL (group 5). To day 30, after rCCH and cell therapy performance, the males from this group displayed signifi cantly higher rates of sexual motivation than in group 2, i. e. the time of barrier examination and approach number to it were 178.57 and 100% higher, respectively, wherein the stimulated Ts level exceeded the corresponding comparison index by 209% (Table 1).
Thus, a combined application of rCCH and cCBL ensured an increase in manifestation rate of sexual activation and Ts level in blood serum of male rats with CAI after exposure to a receptive female.
Some authors considered [6,16] the analysis of seminal fl uid of male rats as able to refl ect the severity of pathological alterations in sexual sphere. Quantitative and qualitative changes in ejaculate parameters in rats could be a crucial criterion for adaptation and maladaptation, occurring in a body when aff ected by various therapeutic measures.
Either rCCH or cCBL administration significantly altered all the studied parameters of seminal fl uid in rats. A combined use of rCCH and cCBL demonstrated the advantages over the application of these methods solely as an independent exposure ( Table 2). For example, the group 4 demonstrated the signifi cant changes in the indices (compared with groups 2 and 3), which characterized the spermatozoa motility and their morphological integrity. This can be interpreted as a positive dynamics in reducing a toxic alcohol eff ect on reproductive function in male rats.
These fi ndings implied that the sperm analysis in male rats could refl ect the severity of pathological shifts in sexual sphere under CAI and serve as a signifi cant criterion for the processes, occurring in a body under alcohol, rCCH and cCBL impacts.
Thus, a combined use of rCCH and cCBL ensured an increase in all the studied indices of reproductive activity in male rats with CAI, showing the enhancement of their sexual activity and Ts level in blood serum following exposure to a receptive female, and a considerable improvement of sperm quality in ejaculate, collected from experimental animals.
To date, the recognizing of alcoholism as a brain disease is undeniable. Like other chronic diseases, it manifests with a complex of behavioral disorders, resulting from interaction of genetic, biological, psychosocial factors and environmental impacts [17,20,24]. Chronic alcohol intoxication provokes a constant additional release of neurotransmitters, causes their functional defi ciency, threatens the body's vital functions and as a result, entails the development of deep impairments in emotional sphere, memory and behavior.
The sexual dysfunction under CAI eff ect we determined during the experiment, are also specifi c to men. Some authors [22,23] described the manifestations of low libido and erectile dysfunction up to the introitus failure together with a sharp decrease in Ts level under alcohol abuse. These effects might be a result of impact of ethanol and various impurities in alcoholic beverages on 'pretesticular' regulatory level of reproductive system.

Висновки
1. Під впливом хронічної алкогольної інтоксикації у експериментальних щурів значуще зни-response to cold exposure not only through the thermoregulatory system, but via all the possible adaptive mechanisms as well, including the hypothalamic-pituitary-adrenal, immune and endocrine systems. The most important aspects of cold physiological eff ect in rCCH include the changes in activity of the higher autonomic centers and neuroendocrine regulation, directly responsible for body's temperature hemostasis [11,12].
Considering the numerous reports and clinical studies [4,19], of great interest are the cord blood cells and possibilities for their clinical application. In particular, previously, we have shown the cCBL impact on correction of CNS and brain structures in the CAI-addicted rats. Signifi cant advantages of combining the rCCH and cCBL administration were also noted [2].
Physiological features of body and its functional systems' responses to rCCH and cCBL administration suggest not only their independent use, but the combination as well. In this case, a mutual potentiation of the eff ect may be achieved, if taking into account the scope and characteristics of each exposure, as well as the structure of pathological process.
So, in this study, we can assume a combined use of rCCH and cCBL to activate the hypothalamic-pituitary system, i. e. to increase the gonadotropin-releasing hormone secretion and release of follicle-stimulating and luteinizing hormones into blood. This resulted in the Ts synthesis activation, enhancement of Sertoli cells metabolism and spermatogenesis stimulation.
Thus, the combination of rCCH and cCBL has a great potential and prospects to correct the pathological states of diff erent genesis and severity.

Conclusions
1. Under chronic alcohol intoxication, the experimental rats revealed a signifi cant decrease in the baseline and stimulated testosterone levels (by 46.1 and 73.81%, respectively), and in total spermatozoa number as well (by 59%), while a number of morphologically defective and dead spermatozoa in ejaculate was increased (by 40 and 31.9%, respectively), thereby signifi cantly reduced the manifestation rate of sexual activation.
sure to a receptive female (by 209%), and the sperm quality improvement in ejaculate (total cell number and percentage of motile cells increased by 91.3 and 44.2%, respectively).