Scrotal/Testicular Thermoregulation in Bulls

Summary

Testicular temperature in bulls must be 2 to 6 degrees Celsius cooler than core body temperature for the production of fertile spermatozoa. Mechanisms that cool the testes include the testicular vascular cone (functions as a counter-current heat exchanger), heat loss from the scrotal surface, relaxation of scrotal muscles, scrotal sweating, whole-body responses, and complementary temperature gradients (in the scrotum and testes). The testes operate on the brink of hypoxia. When testicular temperature increases, metabolism increases at a greater rate than blood flow and hence the testes become hypoxic. Therefore, the testes are very susceptible to temperature increases due to endogenous or exogenous factors (e.g. fever, high ambient temperature). As testicular temperature increases, the proportion of defective spermatozoa increases; recovery is dependent upon the nature and duration of the thermal insult.

Introduction

Bull fertility is of paramount importance in cattle production; one bull may be exposed to 20 females under natural service conditions or hundreds of thousands via artificial insemination. Although few bulls are sterile (unable to reproduce), there is a large range in bull fertility, especially in unselected populations [1]. It is well known that testicular temperature in bulls must be 2 to 6 degrees Celsius below core body temperature for the production of fertile spermatozoa and that increases in testicular temperature decrease semen quality [2]. Increased testicular temperature is the underlying cause of infertility in many bulls.

Anatomy and Physiology

Several features contribute to the regulation of testicular temperature. The pampiniform plexus is a complex venous network surrounding the highly coiled testicular artery; the entire structure (venous network and artery) is properly called the "testicular vascular cone" [3]. In the testicular vascular cone, arterial blood is cooled as heat is transferred from the artery to the vein in a classical counter-current heat-transfer system. Furthermore, this is an important site of surface heat loss as the skin overlying the cone is usually the warmest area on the scrotum [4]. Characteristics of the testicular vascular cone and scrotal surface temperature in bulls from 0.5 to 3 years of age have been reported [5].

Scrotal skin is usually thin and relatively hairless. There is an extensive subcutaneous blood and lymphatic system, with blood vessels located superficially, facilitating heat transfer [6]. Smooth muscle in cutaneous arterioles of the scrotum are innervated by sympathetic neurons [7]; stimulation of these neurons causes vasoconstriction [7]. An increase in scrotal temperature causes dilation of these arterioles by direct action of heat and reflex removal of sympathetic vasoconstrictor tone [8].

The scrotal neck is the warmest part of the scrotum [4]; a long, distinct scrotal neck (pendulous scrotum) provides a large area for heat loss and moves the testes away from the abdomen. The tunica dartos, a thin sheet of smooth muscle under the scrotal skin, is controlled by sympathetic nerves and contracts and relaxes in cold and warm environments, respectively [8]. The cremaster muscle also contracts to move the testes closer to the body in cold conditions; however, unlike the tunica dartos, it is a striated muscle and cannot sustain contraction for prolonged intervals [8].

Sweating and whole-body responses contribute to testicular cooling and have been best characterized in sheep. In Merino rams, scrotal sweat glands are larger and produce more sweat than those elsewhere on the body [9]. Similarly, sweat gland density is higher in scrotal skin than any other body region in bulls [10]. Apocrine sweat glands in the scrotum of rams discharge simultaneously; expulsion begins when scrotal surface temperature is about 35.5 degrees Celsius and occurs at a frequency of up to 10 discharges per hour [11]. Whole-body response in rams includes an increase in respiration rate when scrotal surface temperature rises above 35 - 36 degrees Celsius [8]. Furthermore, when scrotal surface temperature in rams reaches 38 - 40 degrees Celsius, respiration becomes very rapid (e.g. 200 breaths per minute], there is peripheral vasodilation and temperatures within the rectum and carotid artery can decline as much as 2 degrees Celsius in 1 hour [12].

Surface and Internal Temperatures

In 16 crossbred beef bulls [13], temperatures were measured at three locations in each testis: top, middle and bottom. Average temperatures (degrees Celsius) at these locations were: 30.4, 29.8 and 28.8 (scrotal surface temperature); 33.3, 33.0 and 32.9 (scrotal subcutaneous temperature); and 34.3, 34.3 and 34.5 (intratesticular temperature). Top-to-bottom temperature gradients were 1.6, 0.4 and -0.2 degrees Celsius for scrotal surface, scrotal subcutaneous and intratesticular temperatures, respectively. Therefore, the temperature gradient was most pronounced on the scrotal surface, small in the scrotal subcutaneous tissues, and absent within the testicular parenchyma. It was subsequently shown that the scrotal surface and testes have opposing, complementary temperature gradients, resulting in a relatively uniform intratesticular temperature [14]. Furthermore, although intratesticular temperature was significantly higher when a testis was within the scrotum compared to when it was exposed, scrotal surface temperature was similar whether or not there was an underlying testis [14]. Therefore, the scrotum has a significant influence on testicular temperature but the testes have a small influence on scrotal temperature.

Scrotal and testicular temperature gradients may be due to vasculature. The scrotum is vascularized from top to bottom. However, the testicular artery (after exiting the ventral aspect of the testicular vascular cone) courses the length of the testis (under the corpus epididymis), reaches the bottom of the testis, and then diverges into multiple branches that spread dorsally and laterally across the surface of the testis before entering the testicular parenchyma [15]. Therefore, the testis is vascularized from the bottom to the top. In a recent study [16] it was shown that blood within the testicular artery has a similar temperature at the top of the testis (below the testicular vascular cone) compared with the bottom of the testis, but was significantly cooler at the point of entry into the testicular parenchyma (intra-arterial temperatures 34.3, 33.4 and 31.7 degrees Celsius, respectively). Consequently, these opposing temperature gradients collectively result in a nearly uniform intratesticular temperature.

In bulls, temperatures of the caput, corpus and cauda epididymis averaged 35.6, 34.6 and 33.1 degrees Celsius, respectively, and the gradient between the caput and the cauda averaged 2.5 degrees Celsius [13]. The temperature of the caput was greater than that of the testicular parenchyma at the top of the testis, probably because the caput is close to the testicular vascular cone. However, the cauda, an important site for sperm storage and maturation, was slightly cooler than the testicular parenchyma.

Sources of Testicular Heat

Testicular blood flow and oxygen uptake were recently measured in eight Angus bulls to determine the importance of blood flow versus metabolism as sources of testicular heat [17]. Blood flow in the testicular artery averaged 12.4 mL per minute. Arterial blood was warmer (39.2 versus 36.9 degrees Celsius, P<0.001) and had a higher percentage of hemoglobin saturated with oxygen than blood in the testicular vein (95.3 versus 42.0% P<0.001). Based on blood flow and hemoglobin saturation, the oxygen used by one testis (1.2 mL per minute) was calculated to produce 5.8 calories of heat per minute, compared to 28.3 calories per minute attributed to blood flow (approximately a five-fold difference).

The testis usually operates on the brink of hypoxia [8]. Increased temperature increases metabolism, with a concurrent need for increased oxygen to sustain aerobic metabolism. However, studies in rams [8] have shown that blood flow changes little in response to increases in testicular temperature and consequently the testes become hypoxic. Increasing blood oxygen saturation is not practical since the blood is nearly completely saturated under normal conditions. Although increasing blood flow would increase the delivery of oxygen, it would also bring considerable additional heat into the testes. Therefore, increasing heat loss from the scrotum would appear to be the most appropriate response.

Evaluation of Scrotal Surface Temperature with Infrared Thermography

Infrared thermograms of the scrotum of bulls with apparently normal scrotal thermoregulation had left-to-right symmetry and were 4 to 6 degrees Celsius warmer at the top of the scrotum than at the bottom [4,18]. More random temperature patterns, often lacking left-to-right symmetry and having localized areas of increased temperature, were interpreted as abnormal thermoregulation of the testes or epididymides. Although bulls with abnormal thermograms usually had poor quality semen [4,18], not every bull with poor quality semen had an abnormal thermogram. In bulls with unilateral orchitis, the scrotal surface temperature was greater over the affected testis compared to the other testis [18]. Scrotal surface temperature in rams [19] was highly correlated with both scrotal subcutaneous temperature and with the temperature of a surrogate testis (water-filled balloon). However, it is now recognized that caution must be exercised when making inferences about intratesticular temperature based on measurement of scrotal surface temperature [13].

Infrared thermography has been used as an adjunct to the standard breeding soundness examination. Thirty yearling beef bulls that were satisfactory on a standard breeding soundness examination, were individually exposed to approximately 18 heifers for a 45-day breeding period [20]. For bulls with a scrotal surface temperature pattern that was classified as normal or questionable, pregnancy rates 80 days after the end of the breeding season were similar (83 versus 85%), but were higher (P<.01) than pregnancy rates for bulls with an abnormal scrotal surface temperature pattern (68%).

Effects of Increased Testicular Temperature

Increased Ambient Temperature

The effect of increased ambient temperature on semen quality has been determined in many studies. In one study, two Guernsey bulls were exposed to 37 degrees Celsius and 81% relative humidity for 12 hours per day for 17 consecutive days [21]. Approximately 30 to 40% of the spermatozoa were morphologically abnormal (mostly coiled tails and detached heads) and the total number of spermatozoa, sperm concentration, and motility decreased profoundly. In another study [22], ambient temperatures of 40 degrees Celsius at a relative humidity of 35 to 45% for as little as 12 hours reduced semen quality. Bos taurus bulls are more susceptible to high ambient temperatures than Bos indicus bulls [22]. Following exposure to high ambient temperatures, decreases in semen quality were less severe, occurred later and recovered more rapidly in crossbred (Bos indicus x Bos taurus) bulls than in purebred Bos taurus bulls [23].

Scrotal Insulation

Insulation of the scrotum (with cloth, wool or other materials) has been frequently used as a model of increased testicular temperature. In one study [24], the scrotum of Bos indicus x Bos taurus bulls was insulated for 48 hours. The nature and time (Day 0 = initiation of insulation) of morphologically abnormal spermatozoa that resulted were: decapitated, Days 6 - 14; abnormal acrosomes, Days 12 - 23; abnormal tails, Days 12 - 23; and protoplasmic droplets, Days 17 - 23. Therefore, scrotal heating affected spermatozoa in the caput epididymis as well as spermatids. Although daily sperm production was not affected, epididymal sperm reserves were reduced by nearly 50% (9.2 billion versus 17.4 billion), particularly in the caput (3.8 vs 6.6 billion) and cauda (3.7 versus 9.5 billion), perhaps due to selective resorption of abnormal spermatozoa in the rete testis and excurrent ducts. In another study [25,26], the scrota of six Holstein bulls was insulated for 48 hours (Day 0 = initiation of insulation). The number of spermatozoa collected was not significantly affected but the proportion of progressively motile spermatozoa decreased from 69% (prior to insulation) to 42% on Day 15. The proportion of normal spermatozoa was not significantly different from Day -6 to Day 9 (80%), decreased abruptly on Day 12 (53%) and reached a nadir on Day 18 (14%). Although there was considerable variation among bulls in the type and proportion of abnormal spermatozoa, specific abnormalities appeared in a consistent chronological sequence: tailless, Days 12 to 15; diadem, Day 18; pyriform and nuclear vacuoles, Day 21; knobbed acrosome, Day 27; and Dag defect, Day 30. When spermatozoa were collected 3-9 d after insulation and examined immediately, their motility and morphology were similar to pre-insulation values [25]. Compared to semen collected prior to insulation, following freezing, thawing and incubation at 37 degrees Celsius for 3 hours [25], there were significant reductions in the proportion of progressively motile spermatozoa (46 versus 31%, respectively) and the proportion of spermatozoa with intact acrosomes (73 versus 63%). Freezing plus post-thaw incubation manifested changes that had occurred in spermatozoa that were in the epididymis at the time of scrotal insulation.

In a recent study [27], scrotal insulation (4 days) and dexamethasone treatment (20 mg per day for 7 days) were used as models of testicular heating and stress, respectively. Some bulls seemed predisposed to produce spermatozoa with a particular abnormality. Pyriform heads, nuclear vacuoles, microcephalic sperm, and abnormal DNA condensation were more common in insulated than dexamethasone-treated bulls. Conversely, dexamethasone treatment resulted in an earlier and more severe effect on epididymal spermatozoa, an earlier and greater increase in distal midpiece reflexes, and an earlier increase in proximal and distal droplets. In general, the types of defective spermatozoa and the time of their detection were similar for the two treatments.

Insulation of the Scrotal Neck

The scrotal neck of five bulls was insulated for 7 days (Days 1 to 8) as a model of bulls with a excessive body condition (that usually have considerable fat in the neck of the scrotum). Spermatozoa within the epididymis or at the acrosome phase during insulation appeared to be most affected [28]. Insulated bulls had twice as many spermatozoa with midpiece defects and four times as many with droplets on Day 5, fewer normal spermatozoa and three times as many with midpiece defects and droplets on Day 8, fewer normal spermatozoa on Days 15 and 18, and more spermatozoa with head defects on Days 18 and 21. Semen quality in insulated bulls had nearly returned to pre-insulation values by Day 35. In a second experiment [28], scrotal subcutaneous temperature increased 2.0, 1.5 and 0.5 degrees Celsius at the top, middle and bottom of the testis, respectively, and intratesticular temperature was 0.9°C higher at the corresponding three locations 48 hours after scrotal neck insulation compared to pre-insulation. Clearly the scrotal neck is an important site of heat loss.

Increased Epididymal Temperature

In the majority of animals, the cauda epididymis is somewhat cooler than the testes [29], facilitating its sperm storage function. Increasing cauda temperature disrupts the normal absorptive and secretory functions, changes the composition (ions and proteins) of the cauda fluid, and increases (approximately three-fold) the rate of sperm passage through the cauda [29]. Consequently, the number of sperm in the first ejaculate declines, with an even more dramatic decline in sperm number in successive ejaculates. In addition, the increased temperature seems to prematurely hasten sperm maturation [29].

Effects of Increased Temperature on Testicular Cells

Although heating seems to affect Sertoli and Leydig cell function, germ cells are the most sensitive to heat [30]. All stages of spermatogenesis are susceptible, with the extent of damage related to the extent and duration of the increased temperature [30]. Spermatocytes in meiotic prophase are killed by heat, whereas spermatozoa that are more mature usually have metabolic and structural abnormalities [31]. Heating the testis usually decreases the proportion of progressively motile and live spermatozoa, and increases the incidence of morphologically abnormal spermatozoa, especially those with defective heads [32]. Although there is considerable variation among bulls in the nature and proportion of defective spermatozoa, the order of appearance of specific defects is relatively consistent [26,27]. Unless spermatogonia are affected, the interval from cessation of heating to restoration of normal spermatozoa in the ejaculate corresponds to the interval from the beginning of differentiation to ejaculation [30]. Even though sperm morphology has returned to normal, their utilization may result in decreased fertilization rates and an increased incidence of embryonic death [33].

Summary of Increased Testicular Temperature

When scrotal/testicular temperature is increased (regardless of the cause), sperm morphology is generally unaffected initially (for an interval corresponding to epididymal transit time) but subsequently declines [32]. In some studies [24,28], spermatozoa that would have been in the epididymis at the time of scrotal heating were morphologically abnormal when collected soon after heating. In another study [25], changes in these spermatozoa were manifest only after they were frozen, thawed and incubated. Sperm morphology usually returns to pre-treatment values within approximately 6 weeks of the thermal insult. However, a prolonged and (or) severe increase in testicular temperature will increase the interval for recovery. It appears that the decrease in semen quality associated with increased testicular temperature is ultimately related to the severity and the duration of the increased testicular temperature.