Climatic seasons and time of the day influence thermoregulation and testicular hemodynamics in Santa Inês rams raised under humid tropical conditions.

This study evaluated the possible association between the diurnal variations of climatic factors during the rainy (RS) or less rainy (LS) seasons on the testicular hemodynamics and thermoregulatory responses of hair sheep rams raised in a humid tropical climate. Santa Inês rams (n = 6) underwent evaluation of general and testicular physiological parameters (heart and respiratory rates, internal and scrotal temperatures, internal-scrotal temperature gradient, scrotal distention, and color Doppler ultrasound evaluation of the spermatic cords and spectral analyses of testicular arteries) over six consecutive weeks per season at three separate times daily (morning = 8:00 a.m., noon = 12:00 p.m., and afternoon = 5:00 p.m.) during the RS and LS. Climatic air temperature and relative humidity data were recorded, and the temperature and humidity index (THI) was calculated. Higher thermal challenge was observed in LS relative to RS (air temperature = 28.0 vs. 30.9 °C; relative humidity = 84.1 vs. 69.9%; THI = 80.0 vs. 82.5; P < 0.05). In both seasons, respiratory rate and internal temperature were normal, demonstrating the animals' adaptability. In RS, however, a higher scrotal temperature was recorded in relation to LS (35.0 vs. 34.7 °C; P < 0.05), with a gradual increase from morning to afternoon. Lower resistivity (0.40 vs. 0.64; P < 0.05) and pulsatility (0.55 vs. 1.14; P < 0.05) indices, and a higher rate of high-velocity blood flow of testicular arteries (71.1 vs. 60.6%; P < 0.05) were observed in RS compared to LS. The lowest correlations between testicular hemodynamic, physiological variables, and environmental parameters (P < 0.05) were observed in the morning. In conclusion, testicular thermoregulation and testicular hemodynamics were influenced by the climatic seasons and time of the day, being more efficient in the LS season and with less interference from environmental factors in the morning.

Testicular shape, scrotal skin thickness and testicular artery blood flow changes in bulls of different ages.

The aim of this study was to compare the biometric testicular characteristics, skin thickness and haemodynamics of the testicular artery of 12- and 24-month-old bulls using Doppler ultrasonography, the study was conducted using 48 indicus-taurus animals. The scrotal circumference (SC) and biometry characteristics of the bulls were measured to calculate the testicular volume. Doppler ultrasonography was used to obtain the haemodynamic values of the testicular artery. The skin thickness and volume were lower (p<.01) in the younger bulls (12 months:4.68 ± 0.68 mm; 168.76 ± 47.96 cm3 ) versus 24 months (5.05 ± 0.89; 499.73 ± 129.24 cm3 ) animals (p<.01). During diastole, mean velocity was lower in the 12 months (7.98 ± 3.83) than in the 24 months (11.37 ± 4.15) animals (p <.05). The 12-month-old animals had higher pulsatility and resistivity indices (0.49 ± 0.02; 0.51 ± 0.20) compared to the 24-month-old animals (0.32 ± 0.16; 0.40 ± 0.15) (p < .05). The final testicular end velocity was lower in animals with long/moderate-shaped (L/M) (7.31 ± 2.91) than in those moderate/oval-shaped (M/O) (11.48 ± 3.88) testicles (p < .05). Animals with L/M testes presented higher pulsatility values and resistivity indices (0.51 ± 0.05; 0.55 ± 0.04) compared to animals with M/O shape (0.29 ± 0.20; 0.36 ± 0.15). We showed that the blood flow of the supra testicular artery between the two evaluated ages differed, and that 24-month-old bulls presented better thermoregulation capacity. Animals with a long/moderate testicular format presented a greater vascular resistance, which was imposed on the blood flow due to the anatomical differences in the testicular artery, resulting in lower velocity, and indicating better heat dissipation in this format.

Infrared thermography as a noninvasive method to assess scrotal insulation on sperm production in beef bulls.

This study evaluated the thermoregulation and spermatogenic changes by scrotal temperature gradient using infrared thermography in testicular compromised bulls. Bulls were insulated (n = 6) for 72 hr and control animals (n = 3) remained without insulation during all the experimental period. Seminal evaluation was performed prior, at insult removal and once per week for 13 consecutive weeks. Mean temperature gradient in insulated animals was lower at the time of insulation removal compared to the week prior and after the insult (p < .05). Two weeks after insult, sperm motility was lower in insulated compared to control animals (p < .01) and spermatozoa total defects were higher in insulated compared to control animals (p < .05). Two and seven weeks after insult, the major defects were higher in insulated compared to control animals (p < .05). Scrotal temperature gradient showed a positive correlation with sperm mass motion (p < .01) and a negative correlation with ocular globe temperature (p < .01) in insulated animals. The infrared thermography can be used to evaluate ocular globe temperature in bulls; however, it is only effective to detect changes in scrotal temperature gradient at the insult removal.

Measurement of bovine body and scrotal temperature using implanted temperature sensitive radio transmitters, data loggers and infrared thermography.

Synchronous and continuous measurement of body (BT) and scrotal temperature (ST) without adverse welfare or behavioural interference is essential for understanding thermoregulation of the bull testis. This study compared three technologies for their efficacy for long-term measurement of the relationship between BT and ST by means of (1) temperature sensitive radio transmitters (RT), (2) data loggers (DL) and (3) infrared imaging (IRI). After an initial pilot study on two bulls to establish a surgical protocol, RTs and DLs were implanted into the flank and mid-scrotum of six Wagyu bulls for between 29 and 49 days. RT frequencies were scanned every 15 min, whilst DLs logged every 30 min. Infrared imaging of the body (flank) and scrotum of each bull was recorded hourly for one 24-h period and compared to RT and DL data. After a series of subsequent heat stress studies, bulls were castrated and testicular tissue samples processed for evidence of histopathology. Radio transmitters were less reliable than DLs; RTs lost >11 % of data, whilst 11 of the 12 DLs had 0 % data loss. IRI was only interpretable in 35.8 % of images recorded. Pearson correlations between DL and RT were strong for both BT (r > 0.94, P < 0.001) and ST (r > 0.80, P < 0.001). Surgery produced temporary minor inflammation and scrotal hematoma in two animals post-surgery. Whilst scar tissue was observed at all surgical sutured sites when bulls were castrated, there was no evidence of testicular adhesion and normal active spermatogenesis was observed in six of the eight implanted testicles. There was no significant correlation of IRI with either DL or RT. We conclude that DLs provided to be a reliable continuous source of data for synchronous measurement of BT and ST.

Changes in miRNA levels of sperm and small extracellular vesicles of seminal plasma are associated with transient scrotal heat stress in bulls.

Scrotal heat stress affects spermatogenesis and impairs male fertility by increasing sperm morphological abnormalities, oxidative stress and DNA fragmentation. While sperm morpho-functional changes triggered by scrotal heat stress are well described, sperm molecular alterations remain unknown. Recently, spermatozoa were described as accumulating miRNAs during the last steps of spermatogenesis and through epididymis transit, mainly by communication with small extracellular vesicles (sEVs). Herein, the aim was to investigate the impact of scrotal heat stress in miRNAs profile of sperm, as well as, seminal plasma sEVs. Six Nelore bulls (Bos indicus) were divided into two groups: Control (CON; n = 3) and Scrotal Heat Stress (SHS; n = 3; scrotal heat stressed during 96 h by scrotal bags). The day that the scrotal bags were removed from SHS group was considered as D0 (Day zero). Seminal plasma sEVs were isolated from semen samples collected seven days after heat stress (D+7) to evaluate sEVs diameter, concentration, and 380 miRNA levels. Sperm morpho-functional features and profile of 380 miRNAs were evaluated from semen collected 21 days after heat stress (D+21). As a control, sEVs and sperm were analyzed seven days before heat stress (D-7). Only semen parameters that were not significantly different (P > 0.05) among bulls on D-7 were addressed on D+7 and D+21. While no alterations in diameter and concentration were detected in sEVs on D+7 between CON and SHS groups, three sEVs-miRNAs (miR-23b-5p, -489 and -1248) were down-regulated in SHS bulls compared to CON on D+7; other three (miR-126-5p, -656 and -1307) displayed a tendency (0.05 < P < 0.10) to be altered. Sperm oxidative stress was higher, and the level of 21 sperm miRNAs was altered (18 down-, 3 up-regulated) in SHS bulls compared to CON on D+21. Functional analysis indicated that target genes involved in transcription activation, as well as cell proliferation and differentiation were related to the 18 down-regulated sperm miRNAs (miR-9-5p, -15a, -18a, -20b, -30a-5p, -30b-5p, -30d, -30e-5p -34b, -34c, -106b, -126-5p, -146a, -191, -192, -200b, -335 and -449a). Thus, the scrotal heat stress probably impacted testicular and epididymis functions by reducing the levels of a substantial proportion of sEVs and sperm miRNAs. Our findings suggest that miR-126-5p was possibly trafficked between sEVs and sperm and provide new insights on the mechanism by which sperm acquire miRNAs in the last stages of spermatogenesis and sperm maturation in cattle.

Short-term testicular warming under anesthesia causes similar increases in testicular blood flow in Bos taurus versus Bos indicus bulls, but no apparent hypoxia.

Bull testes must be 4-5 °C below body temperature, with testicular warming more likely to cause poor-quality sperm in Bos taurus (European/British) versus Bos indicus (Indian/zebu) bulls. Despite a long-standing dogma that testicular hyperthermia causes hypoxia, we reported that increasing testicular temperature in bulls and rams enhanced testicular blood flow and O2 delivery/uptake, without hypoxia. Our objective was to determine 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. 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. Hypotheses that: 1) both breeds increase testicular blood flow in response to testicular warming; and 2) neither breed has testicular hypoxia, were supported; however, the hypothesis that the relative increase in blood flow is greater in Angus versus Nelore was not supported. Although these were short-term increases in testicular temperature in anesthetized bulls, results did not support the long-standing dogma that increased testicular temperature does not increase testicular blood flow and an ensuing hypoxia is responsible for decreases in motile, morphologically normal and fertile sperm.

Amelioration of heat stress-induced damage to testes and sperm quality.

Heat stress (HS) occurs when temperatures exceed a physiological range, overwhelming compensatory mechanisms. Most mammalian testes are ∼4-5 °C cooler than core body temperature. Systemic HS or localized warming of the testes affects all types of testicular cells, although germ cells are more sensitive than either Sertoli or Leydig cells. Increased testicular temperature has deleterious effects on sperm motility, morphology and fertility, with effects related to extent and duration of the increase. The major consequence of HS on testis is destruction of germ cells by apoptosis, with pachytene spermatocytes, spermatids and epididymal sperm being the most susceptible. In addition to the involvement of various transcription factors, HS triggers production of reactive oxygen species (ROS), which cause apoptosis of germ cells and DNA damage. Effects of HS on testes can be placed in three categories: testicular cells, sperm quality, and ability of sperm to fertilize oocytes and support development. Various substances have been given to animals, or added to semen, in attempts to ameliorate heat stress-induced damage to testes and sperm. They have been divided into various groups according to their composition or activity, as follows: amino acids, antibiotics, antioxidant cocktails, enzyme inhibitors, hormones, minerals, naturally produced substances, phenolic compounds, traditional herbal medicines, and vitamins. Herein, we summarized those substances according to their actions to mitigate HS' three main mechanisms: oxidative stress, germ cell apoptosis, and sperm quality deterioration and testicular damage. The most promising approaches are to use substances that overcome these mechanisms, namely reducing testicular oxidative stress, reducing or preventing apoptosis and promoting recovery of testicular tissue and restoring sperm quality. Although some of these products have considerable promise, further studies are needed to clarify their ability to preserve or restore fertility following HS; these may include more advanced sperm analysis techniques, e.g. sperm epigenome or proteome, or direct assessment of fertilization and development, including in vitro fertilization or breeding data (either natural service or artificial insemination).

A new paradigm regarding testicular thermoregulation in ruminants?

Increased testicular temperature reduces percentages of morphologically normal and motile sperm and fertility. Specific sperm defects appear at consistent intervals after testicular hyperthermia, with degree and duration of changes related to intensity and duration of the thermal insult. Regarding pathogenesis of testicular hyperthermia on sperm quality and fertility, there is a long-standing paradigm that: 1) testes operate near hypoxia; 2) blood flow to the testes does not increase in response to increased testicular temperature; and 3) an ensuing hypoxia is the underlying cause of heat-induced changes in sperm morphology and function. There are very limited experimental data to support this paradigm, but we have data that refute it. In 2 × 3 factorial studies, mice and rams were exposed to two testicular temperatures (normal and increased) and three concentrations of O2 in inspired air (hyperoxia, normoxia and hypoxia). As expected, increased testicular temperature had deleterious effects on sperm motility and morphology; however, hyperoxia did not prevent these changes nor did hypoxia replicate them. In two follow-up experiments, anesthetized rams were sequentially exposed to: 1) three O2 concentrations (100, 21 and 13% O2); or 2) three testicular temperatures (33, 37 and 40 °C). As O2, decreased, testis maintained O2 delivery and uptake by increasing testicular blood flow and O2 extraction, with no indication of anaerobic metabolism. Furthermore, as testicular temperature increased, testicular metabolic rate nearly doubled, but increased blood flow and O2 extraction prevented testicular hypoxia and anaerobic metabolism. In conclusion, our data, in combination with other reports, challenged the paradigm that testicular hyperthermia fails to increase testicular blood flow and the ensuing hypoxia disrupts spermatogenesis.

Acute mild heat stress alters gene expression in testes and reduces sperm quality in mice.

Heat stress is a major concern in animal reproduction, as testicular temperature must be 3-5 °C below body core temperature for production of motile and fertile sperm in mammals. Although recent studies concluded that increased temperature per se was the underlying pathophysiology of testicular impairment, more studies are required to better understand the mechanisms. Therefore, our objective was to investigate the impacts of mild acute heat stress on sperm and testes, and based on mRNA, elucidate involvement of StAR, Trp53 and Trp53-dependent intrinsic and extrinsic apoptotic pathways in pathophysiology of testicular heat stress. Forty-eight C57 BCL6 elite male mice were equally allocated into six groups, anesthetized and the distal third of their body immersed in a water-bath at 40 or 30 °C (heat treatment and control, respectively) for 20 min. Intervals from heat exposure (Day 0) to euthanasia were: 8 and 24 h and 7, 14 and 21 d (plus a control group at 14 d). The epididymides were excised, minced and placed in Tyrode albumin lactate pyruvate hepes (TALPH) at 37 °C for 15 min to recover sperm. Based on computer assisted sperm analysis (CASA), heat treatment reduced total and progressive motility ∼40% (P < 0.05) on Days 14 and 21. Furthermore, percentage morphologically normal sperm was significantly decreased on Day 7, with greater reductions on Days 14 and 21, mostly due to increased midpiece defects. Acrosome integrity (FITC PSA) was decreased ∼35% at 8 h (P < 0.05) and reached a nadir on Day 14. There were decreases (P < 0.05) in seminiferous tubule diameter and testicular weight (relative to body weight) on Day 14. Testicular RNA was extracted, reverse-transcribed and cDNA used for PCR. Expression of genes Hspa1b (Hsp70) and Gpx1 had 7- and 10-fold increases (P < 0.001 for each) at 8 and 24 h, respectively, with Hspa1b remaining upregulated at 24 h, whereas StAR peaked at Day 14 (15-fold, P < 0.0001) and had returned to baseline on Day 21. Both Trp53 and Casp8 were upregulated (P < 0.05) on Day 14, whereas Bcl-2 was decreased (P < 0.05) on Days 7 and 14. In conclusion, acute mild heat stress severely reduced sperm quality and based on mRNA, there was upregulation of chaperone and antioxidant systems and Trp53-dependent intrinsic and extrinsic apoptotic pathways, with deleterious effects on sperm, spermatocytes and spermatids. These findings provided insights into the pathophysiology of heat stress and should contribute to development of evidence-based approaches to mitigate effects of testicular heating.

Testicular hyperthermia reduces testosterone concentrations and alters gene expression in testes of Nelore bulls.

Increased testicular temperature reduces sperm motility, morphology and fertility. Our objectives were to characterize effects of testicular hyperthermia (scrotal insulation) on acute testosterone concentrations and gene expression in Bos indicus testes. Nelore bulls (n = 20), ∼27 mo of age, 375 kg, scrotal circumference >31 cm, with ≥30% motile sperm, were allocated into four groups (n = 5/group): non-insulated (Control) and insulation removed after 12, 24, or 48 h. Immediately after insulation, intratesticular temperatures (needle thermocouples) were coolest in Control bulls and warmest in 48-h bulls (mean ± SEM, 35.28 ± 0.31 vs 38.62 ± 0.57 °C, P < 0.05). Bulls were castrated and testes recovered. Testicular testosterone concentrations were higher in Control versus 48-h bulls (3119 ± 973.3 and 295.5 ± 122.8 ng/g of tissue, respectively, P < 0.05). Total RNA was extracted, reverse transcribed and RT-qPCR done. For STAR, mRNA abundance decreased from Control to 48 h (1.14 + 0.32 vs 0.32 + 0.5, P < 0.05). For BCL2, expression decreased from Control to 24 h (1.00 + 0.07 vs 0.70 + 0.12, P < 0.05), but then rebounded. In addition, GPX1 had a 70% increase (P < 0.05) at 48 h, whereas HSP70 had a 34-fold increase (P < 0.05) at 12 h and 2- and 14-fold increases (P < 0.05) at 24 and 48 h, respectively. HSF1, BAX, P53 and CASP 8 remained unchanged. Downregulation of STAR, critical in androgen production, was consistent with reduced testosterone concentrations, whereas increased GPX1 enhanced testicular antioxidative capability. Huge increases in HSP70 conferred protection again apoptosis and cell destruction, whereas reduced BCL2 promoted apoptosis. These findings provided novel insights into acute tissue responses (testosterone and gene activity) to testicular hyperthermia in B. indicus bulls.

Differences in the thermal sensitivity and seminal quality of distinct ovine genotypes raised in tropical conditions.

For different ovine breeds to maximize their reproductive capacity in countries with tropical climate, it is important to evaluate their potential for thermal resilience and consequences on their reproductive traits. Therefore, the objective of this study was to evaluate the effect of thermal environment temperatures of climate seasons in a tropical climate region on the surface temperatures of the scrotum, testicular biometric characteristics, seminal quality and serum testosterone concentration of rams of different genotypes. Breeders of four different genotypes (Dorper, n = 8, Texel, n = 8, Santa Inês, n = 9 and Morada Nova, n = 8) were used throughout the four climate seasons. Higher thermal challenge was recorded in the spring and summer. In the summer increase in scrotal surface temperature was detected by infrared thermography (P < 0.05), mainly in the regions of the distal testicular pole and tail of the epididymis. The animals of the Texel genotype had higher rectal temperature in the summer. In spring, this genotype also had the highest testicular pole (32.2 ±â€¯0.5 °C; P < 0.05) and distal (29.9 ±â€¯0.4 °C; P < 0.05) temperatures and a higher mean testicular temperature (31.7 ±â€¯0.4 °C; P < 0.05). The Morada Nova genotype showed a higher surface temperature gradient between testicular poles (2.96 ±â€¯0.1 °C; P < 0.05), especially in spring. Genotype-dependent thermal sensitivity was detected for the thermal gradient between the testicular poles, reflecting the seminal quality. There was a positive correlation of the thermal gradient between testicular poles with sperm membrane integrity and negative correlation with total sperm defects. The Texel genotype showed less progressive motility and higher percentage of sperm defects. There was no difference in testosterone concentration between genotypes and in the different seasons (P > 0.05). Thus, the indigenous genotypes showed a greater capability to maintain the scrotum-testicular thermoregulation. Dorper animals resembled the indigenous sheep genotypes, in terms of seminal characteristics, unlike Texel animals, which showed lower adaptability and lower seminal quality.

Review: Testicular vascular cone development and its association with scrotal thermoregulation, semen quality and sperm production in bulls.

Several structural and functional features keep bull testes 2°C to 6°C below body temperature, essential for the production of morphologically normal, motile and fertile sperm. The testicular vascular cone (TVC), located above the testis, consists of a highly coiled testicular artery surrounded by a complex network of small veins (pampiniform plexus). The TVC functions as a counter-current heat exchanger to transfer heat from the testicular artery to the testicular vein, cooling blood before it enters the testis. Bulls with increased TVC diameter or decreased distance between arterial and venous blood, have a greater percentage of morphologically normal sperm. Both the scrotum and testes are warmest at the origin of their blood supply (top of scrotum and bottom of testis), but they are cooler distal to that point. In situ, these opposing temperature gradients result in a nearly uniform testicular temperature (top to bottom), cooler than body temperature. The major source of testicular heat is blood flow, not testicular metabolism. High ambient temperatures have less deleterious effects on spermatogenesis in Bos indicus v. Bos taurus bulls; differences in TVC morphology in B. indicus bulls confer a better testicular blood supply and promote heat transfer. There is a long-standing paradigm that testes operate on the brink of hypoxia, increased testicular temperature does not increase blood flow, and the resulting hypoxia reduces morphologically normal and motile sperm following testicular hyperthermia. However, in recent studies in rams, either systemic hypoxia or increased testicular temperature increased testicular blood flow and there were sufficient increases in oxygen uptake to prevent tissue hypoxia. Therefore, effects of increased testicular temperature were attributed to testicular temperature per se and not to secondary hypoxia. There are many causes of increased testicular temperature, including high ambient temperatures, fever, increased recumbency, high-energy diets, or experimental insulation of the scrotum or the scrotal neck. It is well known that increased testicular temperatures have adverse effects on spermatogenesis. Heat affects all germ cells and all stages of spermatogenesis, with substantial increases in temperature and/or extended intervals of increased testicular temperature having the most profound effects. Increased testicular temperature has adverse effects on percentages of motile, live and morphologically normal sperm. In particular, increased testicular temperature increases the percentage of sperm with abnormal morphology, particularly head defects. Despite differences among bulls in the kind and percentage of abnormal sperm, the interval from increased testicular temperature to the emergence of specific sperm defects is consistent and predictable. Scrotal surface temperatures and structural characteristics of the testis and TVC can be assessed with IR thermography and ultrasonography, respectively.

Arterial blood flow is the main source of testicular heat in bulls and higher ambient temperatures significantly increase testicular blood flow.

Two experiments were done in bulls to determine: total testicular blood flow, testis oxygenation and heat, and effects of ambient temperature on testicular temperatures and blood flow. In Experiment 1, arterial blood flow to testes and testicular oxygenation and heat were determined in Angus bulls (n = 8). Blood temperature and hemoglobin O2 saturation were both greater (P < 0.0001) in the testicular artery than in the testicular vein (39.2 ±â€¯0.2 vs 36.9 ±â€¯0.4 °C and 95.3 ±â€¯0.7 vs 42.0 ±â€¯5.8%, respectively; mean ±â€¯SEM). Based on testicular blood flow of 12.4 ±â€¯1.1 mL/min and an arterial-venous temperature differential of 2.3 °C, blood contributed 28.3 ±â€¯5.1 cal/min of heat to the testis, whereas heat produced by testicular metabolism was estimated at 5.8 ±â€¯0.8 cal/min (based on O2 consumption of 1.2 ±â€¯0.2 mL/min). In Experiment 2, effects of three ambient temperatures (5, 15 and 35 °C) on testicular blood flow and temperatures were determined in 20 Angus bulls. At 35 versus 5 °C, there was greater testicular blood flow (8.2 ±â€¯0.9 versus 4.9 ±â€¯0.7 mL/min/100 g of testicular tissue, P < 0.05), and higher scrotal subcutaneous and intratesticular temperatures (P < 0.01). In conclusion, arterial blood flow was the main source of testicular heat, testes were close to hypoxia, and increased ambient temperature significantly increased scrotal subcutaneous and intratesticular temperatures, as well as testicular blood flow. These studies gave new insights into scrotal/testicular thermoregulation in bulls; they confirmed that testes are nearly hypoxic, but challenged the long-standing paradigm that testicular blood flow does not increase when testes become warmer.