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.

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.

Testicular hyperthermia increases blood flow that maintains aerobic metabolism in rams.

There is a paradigm that testicular hyperthermia fails to increase testicular blood flow and that an ensuing hypoxia impairs spermatogenesis. However, in our previous studies, decreases in normal and motile spermatozoa after testicular warming were neither prevented by concurrent hyperoxia nor replicated by hypoxia. The objective of the present study was to determine the effects of increasing testicular temperature on testicular blood flow and O2 delivery and uptake and to detect evidence of anaerobic metabolism. Under general anaesthesia, the testicular temperature of nine crossbred rams was sequentially maintained at ~33°C, 37°C and 40°C (±0.5°C; 45min per temperature). As testicular temperature increased from 33°C to 40°C there were increases in testicular blood flow (13.2±2.7 vs 17.7±3.2mLmin-1 per 100g of testes, mean±s.e.m.; P<0.05), O2 extraction (31.2±5.0 vs 47.3±3.1%; P<0.0001) and O2 consumption (0.35±0.04 vs 0.64±0.06mLmin-1 per 100g of testes; P<0.0001). There was no evidence of anaerobic metabolism, based on a lack of change in lactate, pH, HCO3- and base excess. In conclusion, these data challenge the paradigm regarding scrotal-testicular thermoregulation, as acute testicular hyperthermia increased blood flow and tended to increase O2 delivery and uptake, with no indication of hypoxia or anaerobic metabolism.

Increased testicular blood flow maintains oxygen delivery and avoids testicular hypoxia in response to reduced oxygen content in inspired air.

Despite a long-standing assertion that mammalian testes operate near hypoxia and increased testicular temperature causes frank hypoxia, we have preliminary evidence that changes are due to hyperthermia per se. The objective was to determine how variations in inspired oxygen concentration affected testicular blood flow, oxygen delivery and extraction, testicular temperature and lactate production. Eight rams were maintained under general anesthesia, with successive decreases in oxygen concentration in inspired air (100, 21 and 13%, respectively). As oxygen concentration decreased from 100 to 13%, there were increases in testicular blood flow (9.6 ± 1.7 vs 12.9 ± 1.9 ml/min/100 g of testis, P < 0.05; mean ± SEM) and conductance (normalized flow; 0.46 ± 0.07 to 1.28 ± 0.19 ml/min/mm Hg/100 g testis (P < 0.05). Increased testicular blood flow maintained oxygen delivery and increased testicular temperature by ~1 °C; this increase was correlated to increased testicular blood flow (r = 0.35, P < 0.0001). Furthermore, oxygen utilization increased concomitantly and there were no significant differences among oxygen concentrations in blood pH, HCO3- or base excess, and no effects of venous-arterial differences in lactate production. In conclusion, under acute hypoxic conditions, testes maintained oxygen delivery and uptake by increasing blood flow and oxygen extraction, with no evidence of anaerobic metabolism. However, additional studies are needed to determine longer-term responses and potential evidence of anaerobic metabolism at the molecular level.

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.

Testis-specific isoform of Na/K-ATPase (ATP1A4) regulates sperm function and fertility in dairy bulls through potential mechanisms involving reactive oxygen species, calcium and actin polymerization.

Traditional bull breeding soundness evaluation (BBSE) eliminates bulls that are grossly abnormal; however, bulls classified as satisfactory potential breeders still vary in field fertility, implying submicroscopic differences in sperm characteristics. The testis-specific isoform of Na/K-ATPase (ATP1A4) is involved in regulation of sperm motility and capacitation in bulls through well-established enzyme activity and signaling functions. The objective was to determine ATP1A4 content, activity and their relationship to post-thaw sperm function and field fertility, using semen samples from low-fertility (LF) and high-fertility (HF) Holstein bulls (n = 20 each) with known FERTSOL rates (measure of field fertility, based on non-return rate). Frozen-thawed sperm from HF bulls had increased ATP1A4 content and activity compared to LF bulls. Furthermore, post-thaw sperm from HF bulls had increased tyrosine phosphorylation, ROS, F-actin content, and low intracellular calcium compared to LF bulls. Subsequent incubation of HF bull sperm with ouabain (a specific ligand of Na/K-ATPase) further augmented the post-thaw increase in tyrosine phosphorylation, ROS production, and F-actin content, whereas the increase in intracellular calcium was still low compared to LF bull sperm. ATP1A4 content and activity, ROS, F-actin and calcium were significantly correlated with fertility. In conclusion, we inferred that ATP1A4 content and activity differed among dairy bulls with satisfactory semen characteristics and that ATP1A4 may regulate sperm function through mechanisms involving ROS, F-actin and calcium in frozen-thawed sperm of HF and LF dairy bulls.

Role of the Na+/K+-ATPase ion pump in male reproduction and embryo development.

Na+/K+-ATPase was one of the first ion pumps studied because of its importance in maintaining osmotic and ionic balances between intracellular and extracellular environments, through the exchange of three Na+ ions out and two K+ ions into a cell. This enzyme, which comprises two main subunits (α and ß), with or without an auxiliary polypeptide (γ), can have specific biochemical properties depending on the expression of associated isoforms (α1ß1 and/or α2ß1) in the cell. In addition to the importance of Na+/K+-ATPase in ensuring the function of many tissues (e.g. brain, heart and kidney), in the reproductive tract this protein is essential for embryo development because of its roles in blastocoel formation and embryo hatching. In the context of male reproduction, the discovery of a very specific subunit (α4), apparently restricted to male germ cells, only expressed after puberty and able to influence sperm function (e.g. motility and capacitation), opened a remarkable field for further investigations regarding sperm biology. Therefore, the present review focuses on the importance of Na+/K+-ATPase on male reproduction and embryo development.

Effect of reproductive seasonality on gamete quality in the North American bison (Bison bison bison).

The objective was to investigate the effects of reproductive seasonality on gamete quality in plains bison (Bison bison bison). Epididymal sperm (n = 61 per season), collected during the breeding season (July-September), had significantly higher post-thaw total motility (36.76 ± 14.18 vs 31.24 ± 12.74%), and lower linearity (0.36 ± 0.06 vs 0.39 ± 0.04) and wobbliness (0.49 ± 0.04 vs 0.51 ± 0.03; mean ± SD) compared to non-breeding season (January-March) samples. Representative samples (n = 4) from each season were used in heterologous IVF trials using cattle oocytes. Cleavage, morulae and blastocyst percentage were higher for breeding vs non-breeding season sperm samples (81.88 ± 6.8 vs 49.94 ± 6.77; 41.89 ± 13.40 vs 27.08 ± 23.21; and 30.49 ± 17.87 vs 13.72 ± 18.98%, respectively). Plains bison ovaries collected during the breeding (n = 97 pairs) and non-breeding (n = 100 pairs) seasons were classified as luteal or follicular. Oocytes recovered from these ovaries were classified into five grades based on morphology. There was no significant difference in the number of luteal ovaries or grades of oocytes recovered. Oocytes were matured, fertilized (with frozen sperm from three bison bulls) and cultured in vitro. Cleavage percentage was higher for oocytes collected during breeding vs non-breeding season (83.72 ± 6.42 vs 73.98 ± 6.43), with no significant difference in subsequent development to blastocysts. In summary, epididymal sperm from non-breeding season had decreased total motility and resulted in reduced embryo production in vitro. Oocytes collected during non-breeding season had reduced ability to be matured, fertilized and/or undergo cleavage in vitro. Data suggested that season influenced gamete quality in plains bison.

The effects of increased testicular temperature on testis-specific isoform of Na+/K+ -ATPase in sperm and its role in spermatogenesis and sperm function.

Impaired testicular thermoregulation is commonly implicated in abnormal spermatogenesis and impaired sperm function in animals and humans, with outcomes ranging from subclinical infertility to sterility. Bovine testes must be maintained 4-5 °C below body-core temperature for normal spermatogenesis. The effects of elevated testicular temperature have been extensively studied in cattle using a scrotal insulation model, which results in abnormal spermatogenesis and impaired sperm morphology and function. Using this model and proteomic approaches, we compared normal and abnormal sperm (from the same bulls) to elucidate the molecular basis of impaired function. We identified a cohort of sperm functional proteins differentially expressed between normal vs abnormal sperm, including a testis-specific isoform of Na(+) /K(+) -ATPase. In addition to its role as a sodium pump regulating sperm motility, Na(+) /K(+) -ATPase is also involved as a signalling molecule during sperm capacitation. In conclusion, because of its involvement in regulation of sperm function, this protein has potential as a fertility marker. Furthermore, comparing normal vs abnormal sperm (induced by scrotal insulation) is a useful model for identifying proteins regulating sperm function.

Moribund sperm in frozen-thawed semen, and sperm motion end points post-thaw and post-swim-up, are related to fertility in Holstein AI bulls.

The objectives were to compare testicular physical characteristics and post-thaw sperm characteristics and their associations with fertility in Holstein bulls used for AI. Ten Holstein bulls (4-5 y old) were classified as either high-fertility (HF) or low-fertility (LF; n = 5 each), based on adjusted 56-d non-return rates [non-return rate (NRR); range (mean ± SD): 55.6 ± 4.6 to 71.8 ± 1.3%). Testicular physical characteristics were not significantly different between the two groups. Four ejaculates were collected from each bull and cryopreserved. Several indexes of sperm motion (based on computer-assisted sperm analysis) at post-thaw and post-swim-up were correlated with NRR. Sperm from HF bulls were in transition to a hyperactivated motility pattern, whereas those from LF bulls had only a forward progressive motility pattern. In HF vs LF bulls, there was a greater percentage of viable sperm after thawing (60.6 ± 9.7 vs 49.5 ± 8.0%, P < 0.05) and after swim-up (70.9 ± 11.0 vs 63.0 ± 8.8%, P < 0.01); these two end points were positively correlated with fertility (r = 0.45, P < 0.01 and r = 0.78; P < 0.01, respectively). Furthermore, in HF vs LF bulls, the ratio of sperm recovered after swim-up to viable sperm in post-thaw semen was higher (P < 0.001), and the proportion of moribund sperm expressed as a percentage of live sperm differed (12.6 ± 3.4 vs. 16.4 ± 3.1%, P < 0.001) and was negatively correlated (r = -0.33, P < 0.05) with fertility. In conclusion, fertility of Holstein bulls maintained in a commercial AI center was not predicted by testicular physical characteristics, but it was associated with differences in moribund sperm in the inseminate, as well as characteristics of sperm post-thaw and after swim-up.

Evaluation of an animal protein-free semen extender for cryopreservation of epididymal sperm from North American bison (Bison bison).

The objective was to evaluate the suitability of an animal protein-free semen extender for cryopreservation of epididymal sperm from the two subspecies of North American bison: plains (Bison bison bison) and wood (Bison bison athabascae) bison. Both cauda epididymides (from six plains and five wood bison) were minced and incubated in Sp-TALPH buffer for approximately 2 h at 37 °C to release actively motile sperm. Sperm suspensions were filtered, centrifuged and the sperm pellet from each bull was divided into two fractions and diluted either in egg yolk containing extender, Triladyl, or in an animal protein-free extender, Andromed, and equilibrated for 20 min at 37 °C. Thereafter, samples were chilled and cryopreserved. Frozen-thawed sperm were evaluated for motility (computer assisted sperm analysis), viability (SYBR 14 and propidium iodide), acrosome integrity (FITC conjugated PSA), cryocapacitation (tyrosine phosphorylation of sperm proteins as a biomarker), and fertilizing ability (in a heterologous IVF system). There was no significant difference for progressive motility, viability, and acrosome integrity between the two extenders for plains bison (36.8 ± 9.0, 60.5 ± 17.4, and 77.3 ± 4.6%; overall mean ± SD) as well as for wood bison (11.7 ± 8.1, 13.7 ± 5.6, and 73.4 ± 4.2%). Levels of tyrosine phosphorylation did not differ for sperm preserved in the two extenders for both subspecies, although an inter-bull variability in the response to tyrosine phosphorylation between extenders was suggested for plains bison. Fertilization percent did not differ significantly between extenders for plains bison (84.16 ± 9.92%, overall mean ± SD) and for wood bison (59.53 ± 19.99%). In conclusion, in the absence of significant difference between extenders in post-thaw sperm characteristics, we inferred that Andromed (animal protein-free) was suitable for cryopreservation of epididymal sperm from North American bison.

Recovery and cryopreservation of epididymal sperm of plains bison (Bison bison bison) as a model for salvaging the genetics of wood bison (Bison bison athabascae).

The objective of this study was to optimize recovery and cryopreservation of epididymal sperm from plains bison, as a model for wood bison. In Phase 1, cauda epididymides were recovered from bison (n = 14) immediately after slaughter, minced and incubated in Sp-TALPH buffer for 3 h at 36 degrees C. The resulting sperm suspensions were cryopreserved in Triladyl, using a protocol for bovine semen. In Phase 2, epididymal sperm were cryopreserved in either Triladyl or Andromed. The mean (+/-SD) estimated number of sperm recovered was 468 +/- 207 x 10(6). There was an increase (p < 0.05) in the proportion of sperm with normal morphology between initial recovery and after extension (52.4 +/- 4.6 vs 69.7 +/- 2.4%), with a concurrent decrease (p < 0.05) in the proportion of sperm with distal droplets. Median values for progressively motile sperm in post-thaw samples (60%) were lower (p < 0.05) than that after extension or after chilling (70% for both). The mean percentages of viable sperm and of sperm with an intact acrosome were lower (p < 0.05) for frozen-thawed samples (38.7 +/- 2.8 and 85.2 +/- 1.1) compared with extended (66.2 +/- 2.2 and 92.4 +/- 0.9) or chilled (63.7 +/- 2.5 and 90.0 +/- 1.0) samples. Rates of cleavage, morulae and blastocyst production were not significantly different for chilled (70.9, 38.7 and 8.0%) vs post-thaw sperm (73.0, 46.0 and 6.3%). There was no significant difference between extenders for most sperm characteristics. In conclusion, we developed a functional protocol for the recovery and cryopreservation of epididymal sperm from plains bison, which may have implications for the genetic preservation of wood bison.

Breeding soundness evaluation and semen analysis for predicting bull fertility.

Bull fertility is influenced by numerous factors. Although 20-40% of bulls in an unselected population may have reduced fertility, few are completely sterile. Breeding soundness refers to a bull's ability to get cows pregnant. A standard breeding soundness evaluation identifies bulls with substantial deficits in fertility, but does not consistently identify sub-fertile bulls. In this regard, the use of frozen-thawed semen (from bulls in commercial AI centres) that meets minimum quality standards can result in pregnancy rates that differ by 20-25 percentage points. Although no single diagnostic test can accurately predict variations in fertility among bulls that are producing apparently normal semen, recent studies suggested that a combination of laboratory tests were predictive of fertility. This review is focused on recent developments in prediction of bull fertility, based on assessments at the molecular, cellular and whole-animal levels.