[19], we exposed 18 rams (nine had an insulated scrotum) to air containing 14, 21, or 85% O2 for approximately 30 hours. In that study, scrotal insulation (to increase testicular temperature) substantially reduced sperm motility (from 58 to 30%) and proportion of morphologically normal sperm (from 87 to 30%), but effects due to O2 were minimal. In a second study [20], 96 male CD‐1 mice were maintained at 20 vs 36 °C, exposed to 13, 21, or 95% O2 twice for 12‐hour intervals (separated by 12 hours at room temperature and 21% O2), and euthanized 14 or 20 days after exposure. Interestingly, sperm morphology and specific stages of sperm cell development were altered in mice exposed to 36 °C, including increases in percentage of sperm with defective heads (P < 0.0001) or tails (P < 0.001) and percentage of altered elongated spermatids (P < 0.001). Regarding effects due to O2 variations, seminiferous tubule diameter and epididymal sperm reserves were reduced in the 13% O2 group, but sperm quality and production were not consistently disrupted by hypoxia. In addition, no hyperthermia‐induced disruptions were prevented by hyperoxia, indicating a major role of increased temperature, but not hypoxia. There were primarily main effects of temperature; mice exposed to 36 °C had smaller testes, fewer morphologically normal sperm, and histologically increased altered spermatids and altered germ cells compared to mice exposed to 20 °C. In both studies, our hypotheses were not supported; sperm quality and production were not consistently disrupted by hypoxia and hyperoxia did not protect against hyperthermia in mice.
We recently conducted two studies in rams under general anesthesia to determine effects of hypoxia and of testicular hyperthermia on testicular blood flow, and O2 delivery and uptake. In the first study [21], eight rams were exposed to successive decreases in O2 concentration in inspired air (100, 21, and 13%; 45 minutes at each concentration). As O2 concentration decreased (100 to 13%), testicular blood flow increased (9.6 vs 12.9 ml/min/100 g of testis, P < 0.05). Increased testicular blood flow maintained O2 delivery and increased testicular temperature by ~1 °C. In the second experiment [22], testicular temperatures of nine crossbred rams were sequentially maintained at 33–35, 37, and 40 °C (45 minutes at each temperature). As testicular temperature increased from 33–35 to 40 °C, there were increases in mean testicular blood flow (9.8 vs 12.2 ml/min/100 g of testes, P < 0.05), O2 extraction (31.2 vs 47.3%, P < 0.0001), and O2 use (0.35 vs 0.64 ml/min/100 g of testes, P < 0.0001). In both experiments, there was no evidence of anaerobic metabolism, based on no significant difference in lactate, pH, HCO3 –, and base excess.
Following our studies in rams, we conducted another study to determine the effects of short‐term testicular hyperthermia on testicular blood flow, O2 delivery and uptake, and evidence of testicular hypoxia in pubertal Angus (B. taurus) and Nelore (B. indicus) bulls (nine per breed) under isoflurane anesthesia [23]. As testes were warmed from 34 to 40 °C, there were increases (P < 0.0001, but no breed effects) in testicular blood flow (mean ± SEM, 9.59 ± 0.10 vs 17.67 ± 0.29 ml/min/100 g, respectively), O2 delivery (1.79 ± 0.06 vs 3.44 ± 0.11 ml O2/min/100 g), and O2 consumption (0.69 ± 0.07 vs 1.25 ± 0.54 ml O2/min/100 g), but no indications of testicular hypoxia (Figure 4.1). Our hypothesis that Angus bulls have a greater relative increase in testicular blood flow than Nelore in response to increased testicular temperature was not supported, as there was no significant breed difference.
Figure 4.1 Mean (and SEM) blood flow, O2 delivery, and metabolic rate in testes of 18 bulls (Nelore and Angus) sequentially exposed to three plateaus of testicular temperature (33‐35, 37, and 40 °C). Assessment of blood flow and sample collection were done four times at 15‐minute intervals and then the testes warmed to reach the next temperature plateau. For each end point, there was a difference (P < 0.001) between all temperature plateaus. Testicular temperature increased testicular metabolism; however, testicular blood flow nearly doubled, providing ample O2 to meet metabolic demands, with no evidence of hypoxia.
Our studies in conscious rams and mice, as well as in anesthetized rams and bulls, challenged the classical paradigm regarding scrotal/testicular thermoregulation, as acute testicular hyperthermia caused increases in blood flow and in delivery and uptake of O2, with no indications of hypoxia. In contrast to the long‐standing paradigm, our data were evidence that effects of increased testicular temperature were due to testicular temperature per se and not secondary hypoxia.
Evaluation of Scrotal Surface Temperature with Infrared Thermography
Infrared thermograms of the scrotum of bulls with apparently normal scrotal thermoregulation were symmetrical left to right, with temperature at the top 4–6 °C warmer than at the bottom [24, 25]. More random temperature patterns, including a lack of horizontal symmetry and areas of increased scrotal surface temperature, were interpreted as abnormal thermoregulation of the testes or epididymides. Nearly all bulls with an abnormal thermogram had reduced semen quality [24, 25]; conversely, not every bull with poor quality semen had an abnormal thermogram. Consequently, infrared thermography is a useful tool for breeding soundness evaluation of bulls, although it does not replace collection and evaluation of semen. In one study, 30 yearling beef bulls, all deemed breeding sound on a standard breeding soundness examination, were individually exposed to approximately 18 heifers for 45 days [26]. Pregnancy rates 80 days after the end of the breeding season were similar (83 vs 85%) for bulls with a normal or questionable scrotal surface temperature pattern, respectively, but were higher (P < 0.01) than pregnancy rates for bulls with an abnormal scrotal surface temperature pattern (68%).
Effects of Increased Testicular Temperature
Increased Ambient Temperature
Effects of increased ambient temperature on semen quality have been widely reported. In one study, ambient temperatures of 40 °C at a relative humidity of 35–45% for 12 hours reduced semen quality [27]. Furthermore, B. taurus bulls are more susceptible than B. indicus bulls to high ambient temperatures [27]. In that regard, decreases in semen quality were less severe, occurred later, and recovered more rapidly in crossbred (B. indicus × B. taurus) bulls than in purebred B. taurus bulls exposed to high ambient temperatures [28].
Scrotal Insulation as a Model of Increased Testicular Temperature
Scrotal insulation is frequently used to increase testicular temperature. In one study [29], scrota of B. indicus × B. taurus bulls were insulated for 48 hours. The nature and time (day 0, start of insulation) of the morphologically abnormal sperm that resulted were as follows: decapitated, days 6–14; abnormal acrosomes, days 12–23; abnormal tails, days 12–23; and protoplasmic droplets, days 17–23. Therefore scrotal heating affected sperm 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 vs 17.4 billion), particularly in the caput (3.8 vs 6.6 billion) and cauda (3.7 vs 9.5 billion), perhaps due to selective resorption of abnormal sperm in the rete testis and excurrent ducts. In another study [30, 31], scrota of six Holstein bulls were insulated for 48 hours (day 0, initiation of insulation). The number of sperm collected was not significantly different, but the proportion of progressively motile sperm decreased from 69% (prior to insulation) to 42% on day 15. The proportion of normal sperm 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 both type and proportion of abnormal sperm, specific abnormalities appeared in a consistent chronological sequence: tailless, days 12–15; diadem, day 18; pyriform