Effects of therapeutic ultrasound and moderate heat on stallion testes

Male infertility causes significant financial losses to horse breeders. Currently, there is no effective treatment for stallion infertility caused by advanced testicular degeneration, where most or all the spermatogonial stem cells (SSCs) are lost from the seminiferous epithelium in the testes. Recent studies have shown that the germ cell pool can be successfully restored and spermatogenesis restarted in the severely degenerated testes of various animal species by the transplantation of SSCs [[1], [2], [3]]. This breakthrough development is now being investigated as a new option for treating infertility in azoospermic men [4,5]. Initial research on the transplantation of testicular cells to the testes was performed on a mouse model [6,7]. Later, similar studies were performed on other laboratory animals, various species of domestic animals, and primates [[8], [9], [10], [11], [12], [13], [14]]. Transplanted spermatogonial, adipose tissue-derived, or bone marrow-derived stem cells entered spermatogenesis, producing donor-derived sperm in male rats, rabbits, dogs, cats, sheep, goats, bulls, pigs, and monkeys [3,11,[14], [15], [16], [17], [18], [19], [20]]. In rats, sheep, and goats, offspring were produced [19,21,22]. To date, evidence of successful intratesticular stem cell transplantation (SCT) in stallions, which has resulted in sperm production and restored fertility, is lacking.

It has been demonstrated in early studies that the availability of an optimal recipient is critical for the success of intratesticular SST [13]. Therefore, prior to performing research studies on the methodology and the effectiveness of intratesticular SCT in horses, an optimal recipient model with severely affected testicular function has to be established. In the most successful studies on laboratory and farm animals, recipients had atrophied testes with a partially or entirely obliterated endogenous germ cell pool. This approach assures a niche for exogenous stem cells, and that donor cells do not have to negotiate multiple layers of the seminiferous epithelium to reach the basement membrane. There are two well-established recipient models for intratesticular SCT: chemotherapy with busulfan, which is known to have strong antispermatogenic properties [23], or testicular irradiation [24]. However, busulfan is a potent cytotoxic drug that sometimes causes severe, even lethal, side effects [25]. Recently, the administration of a high dose of busulfan to three stallions resulted in the death of two of them [26]. Even local busulfan administration into ram lamb testes caused the death of 40% of these animals [27]. Furthermore, working with this drug requires specialized personnel training, isolated animal care facilities, and special waste disposal methods. Irradiation requires specialized and expensive equipment and may lead to calcification in the seminiferous tubules (STs) [28]. More recently, other, safer methods of depleting endogenous germ cells (GCs) from testes were studied. In one of these studies, the scrota of 12 rats were submerged in a 43 °C water bath for 20 min every other day for five weeks, which resulted in the complete GCs obliteration from STs [29]. Furthermore, the authors of this study concluded that testicular hyperthermia is a safer and more effective method in depleting testes from endogenous GCs than busulfan. Several researchers investigated the effects of hyperthermia. induced by scrotal insulation, on stallion testes [[30], [31], [32]]. None of the proposed protocols of scrotal insulation caused severe and long-lasting depletion of the endogenous GCs in stallion testes, however [30,31]. Most likely, scrotal insulation does not increase testicular temperature high enough to cause severe testicular degeneration. Since water bath is not practical in stallions, other methods of increasing scrotal temperature need to be identified.

Several techniques are routinely used to elevate local temperature in injured muscles or joints to alleviate pain and promote healing [[33], [34], [35], [36]]. Heat wraps or patches are readily available to the general public, while a trained physiotherapist usually uses therapeutic ultrasound. While there are no published studies on the effect of heat wraps on testicular function, therapeutic ultrasound (TUS) was investigated as a candidate for a reversible male contraceptive in rats and rhesus monkeys [37,38]. The sustained reduction in sperm numbers, motility, and numbers of morphologically normal sperm was reported after TUS treatment. In addition, repeated applications of TUS in dogs caused severe testicular degeneration, including total loss of spermatogonia from the seminiferous epithelium [39].

To date, there have been no published studies on the effects of moderate heat on the stallion scrotum utilizing heating wraps or TUS. Therefore, we performed a series of “proof of concept” studies aimed at these four goals: 1. to identify an effective TUS device and protocol for increasing intratesticular temperature in stallion testes by at least 5 °C (ex vivo studies); 2. to test the ability of TUS to induce testicular degeneration in stallions (in vivo study); 3. to identify an effective method and protocol to increase the intratesticular temperature to approximately 43 °C in stallion testes and to hold it on this level for approximately 2 h (ex vivo studies); 4. to test the ability of moderate heat alone or in combination with TUS to induce testicular degeneration in stallions (in vivo study).

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