Optimized total RNA isolation from bovine sperm with enhanced sperm head lysis.

Increasing evidence of sperm RNA's role in fertilization and embryonic development has provided impetus for its isolation and thorough characterization. Sperm are considered tough-to-lyse cells due to the compact condensed DNA in sperm heads. Lack of consensus among bovine sperm RNA isolation protocols introduces experimental variability in transcriptome studies. Here, we describe an optimized method for total RNA isolation from bovine sperm using the TRIzol reagent. This study critically investigated the effects of various lysis conditions on sperm RNA isolation. Sperm suspended in TRIzol were subjected to a combination of mechanical treatments (sonication and passage through a 30G needle and syringe) and chemical treatments (supplementation with reducing agents 1,4-dithiothreitol and tris(2-carboxyethyl) phosphine hydrochloride (TCEP)). Microscopic evaluation of sperm lysis confirmed preferential sperm tail versus sperm head lysis. Interestingly, only TCEP-supplemented TRIzol (both mechanical treatments) had progressive sperm head lysis and consistently yielded total sperm RNA. Furthermore, RNA integrity was confirmed based on the electrophoresis profile and an absence of genomic DNA and somatic cells (e.g., epithelial cells, spermatids, etc.) with RT-qPCR. Our findings highlighted the importance of sperm lysis, specifically of the sperm head using TCEP with mechanical treatment, in total RNA isolation and presented a bovine-specific sperm RNA isolation method to reduce experimental variabilities.

Angus bulls voluntarily access shade during hot weather, reducing scrotal subcutaneous temperatures and improving sperm quality.

The objective of this study was to determine the relationship of bull location (shade versus no shade), scrotal subcutaneous and ambient temperatures, and sperm quality. Six Angus bulls (4 to 5 y) were randomly allocated into 2 groups of 3 bulls each, housed in 2 outdoor pens, with 1 containing a shed (~3.5 × 6 m and 2.5 m high, 1 open side) to provide shade. Semen was collected by electroejaculation once weekly for 9 wk. The percentage of time a bull voluntarily accessed shade for ≥ 15 min (observed with a game camera) increased with the ambient temperature and ranged from 7.6% to 86.7% for ambient temperatures of < 25°C and > 33°C, respectively. During the 10 hottest days, scrotal subcutaneous temperature (measured hourly with an implanted data logger) in the bulls without access to shade (control group) was directly associated with ambient temperature. Conversely, bulls with access to shade had lower (P = 0.001) scrotal subcutaneous temperatures during high ambient temperatures, particularly when they accessed shade. During the 4 hottest days, these bulls voluntarily accessed shade most of the time from 12:00 noon to 5:00 p.m. (peak ambient temperatures). For total sperm morphological abnormalities and acrosome integrity, there were group effects (P = 0.001 for each), plus a time effect for acrosome integrity (P = 0.009). For total and progressive forward sperm motility, there were group effects (P = 0.001 and 0.023, respectively). For sperm motility kinetics, which were measured with computer-assisted sperm analysis (CASA), [average path velocity (VAP), curvilinear velocity (VCL), straight line velocity (VSL), straightness of track (STR), and linearity of track (LIN)], there were also group effects (P = 0.005, 0.011, 0.010, 0.020, and 0.046, respectively). In summary, during hot weather, bulls voluntarily accessed shade, which significantly lowered scrotal subcutaneous temperatures and improved sperm quality.

L'objectif de cette étude était de déterminer la relation entre l'emplacement du taureau (à l'ombre ou sans ombre), les températures sous-cutanées et ambiantes du scrotum et la qualité du sperme. Six taureaux Angus (4 à 5 ans) ont été répartis au hasard en deux groupes de trois taureaux chacun, logés dans deux enclos extérieurs, dont un ayant un abri (~3,5 × 6 m et 2,5 m de haut, un côté ouvert) pour fournir de l'ombre. Le sperme a été recueilli par électroéjaculation une fois par semaine pendant 9 semaines. Le pourcentage de temps pendant lequel un taureau a accédé volontairement à l'ombre pendant ≥ 15 min (observé avec une caméra de chasse) augmentait avec la température ambiante et variait de 7,6 % à 86,7 % pour des températures ambiantes de < 25 °C et > 33 °C, respectivement. Pendant les 10 jours les plus chauds, la température sous-cutanée scrotale (mesurée toutes les heures avec un enregistreur de données implanté) chez les taureaux sans accès à l'ombre (groupe témoin) était directement associée à la température ambiante. À l'inverse, les taureaux ayant accès à l'ombre avaient des températures sous-cutanées scrotales plus basses (P = 0,001) lors de températures ambiantes élevées, en particulier lorsqu'ils accédaient à l'ombre. Durant les quatre jours les plus chauds, ces taureaux ont accédé volontairement à l'ombre la plupart du temps de 12h00 à 17h00. (températures ambiantes maximales). Pour les anomalies morphologiques totales des spermatozoïdes et l'intégrité de l'acrosome, il y avait des effets de groupe (P = 0,001 pour chacun), plus un effet de temps pour l'intégrité de l'acrosome (P = 0,009). Pour la motilité totale et progressive des spermatozoïdes, il y avait des effets de groupe (P = 0,001 et 0,023, respectivement). Pour la cinétique de la motilité des spermatozoïdes, qui ont été mesurées avec une analyse de sperme assistée par ordinateur (CASA), [la vitesse moyenne du trajet (VAP), la vitesse curviligne (VCL), la vitesse en ligne droite (VSL), la rectitude de la voie (STR) et la linéarité de la voie (LIN)], il y avait aussi des effets de groupe (P = 0,005, 0,011, 0,010, 0,020 et 0,046, respectivement). En résumé, par temps chaud, les taureaux ont volontairement accédé à l'ombre, ce qui a considérablement abaissé les températures sous-cutanées du scrotum et amélioré la qualité du sperme.(Traduit par Docteur Serge Messier).

Melatonin improves post-thaw sperm quality after mild testicular heat stress in rams.

The objective was to determine effects of slow-release melatonin on post-thaw sperm quality in rams exposed to mild testicular heat stress (HS; scrotal neck insulation). Twelve yearling Dorset rams were randomly and equally allocated to receive either 36 mg melatonin in 1 ml corn oil or 1 ml corn oil injected subcutaneously (SQ); 15 day later, all rams had HS for 96 h (start of HS = start of Week 0). Semen was collected before HS and once weekly from Weeks 1 to 7, extended in Steridyl CSS One Step, held at 5°C for ~3 h, loaded into 0.5 ml straws, held 5 cm above liquid nitrogen for 10 min and then plunged. Computer assisted semen analysis (CASA) was conducted on frozen-thawed sperm. There were group and week effects for total and progressive motility (p < .001), plus group and week effects and group*week interactions (p < .001) for post-thaw total abnormalities, acrosome integrity, post-thaw sperm DNA fragmentation index (DFI) and high mitochondrial membrane potential (HMMP). Post-thaw sperm total and progressive motility, acrosome integrity and HMMP were higher (p < .05) in melatonin versus control groups from Weeks 1 to 7, and the melatonin group reached baseline level (pre-heat stress) at Week 7 (75.79 ± 0.96, 65.48 ± 1.51, 75.00 ± 0.89 and 67.00 ± 1.06, respectively; mean ± SEM). Conversely, post-thaw sperm total abnormalities and DFI were lower (p < .05) in melatonin versus control, and both reached baseline at Week 7 in the melatonin group (26.00 ± 0.57 and 5.66 ± 0.17, respectively). Coiled tails, distal midpiece reflexes, distal cytoplasmic droplets, ruffled acrosomes, bowed midpieces, pyriform heads and knobbed acrosomes were the most common abnormalities in both groups, with lower percentages in melatonin-treated rams. Results supported our hypothesis that HS reduces post-thaw sperm quality, and that melatonin lessens those reductions, manifested by significantly better total and progressive motility, acrosome integrity and HMMP, and fewer sperm total abnormalities and DFI.

Melatonin improves testicular hemodynamics and sperm quality in rams subjected to mild testicular heat stress.

Melatonin is a potent free-radical scavenger, with anti-inflammatory, anti-oxidative, and anti-apoptotic effects. The objective was to determine whether melatonin promoted testicular blood flow and protected sperm quality in rams after mild heat stress (HS; scrotal neck insulation). Twelve yearling Dorset rams with good semen quality were housed indoors (∼18-20 °C). Once weekly for 2 wk, Doppler indices (resistive index [RI] and pulsatility index [PI]) were measured in the supratesticular artery and semen collected by electroejaculation. Then, rams were randomly allocated into two equal groups, and given either 36 mg melatonin in 1 ml corn oil SQ under the ear (MEL), or only corn oil (CONT). At 15 d after treatment, all rams were subjected to mild HS for 96 h, with blood flow measurements and semen collection done once weekly for 7 wk. There were group, week and group∗week interaction effects (P < 0.005) for total and progressive sperm motility (CASA); total sperm abnormalities and acrosome integrity had effects of group, week and group∗week interaction effects (P < 0.00); and there were group and week effects for RI and PI (P < 0.005), with no significant differences before treatment. Changes in total and progressive motility and sperm abnormalities were evident at Week 1 post-HS in CONT rams, but MEL mitigated (P ˂ 0.05) these effects from Weeks 2-7. Furthermore, both PI and RI were reduced (P ˂ 0.05; i.e., significant increase in blood flow) in MEL versus CONT rams most weeks after HS. In MEL rams, sperm motility and total abnormalities had recovered at Weeks 5 and 6, respectively, whereas CONT rams had not completely recovered by Week 7. There was no difference (P < 0.05) between MEL and CONT groups in scrotal subcutaneous temperatures in the 4-d intervals before, during and after HS. In conclusion, melatonin significantly improved testicular blood flow and protected sperm motility and morphology in rams exposed to testicular HS. Therefore, melatonin has potential for mitigating effects of testicular HS under field conditions.

Melatonin or L-arginine in semen extender mitigate reductions in quality of frozen-thawed sperm from heat-stressed rams.

Our objective was to determine effects of melatonin or L-arginine on quality of frozen-thawed sperm from heat-stressed (HS) rams. Ten Dorset rams were randomly allocated to either scrotal neck insulation for 3.5 d or whole-body heating (28 °C and 30-34% RH for 8 h/d for 4 consecutive days). Semen was collected before HS then once weekly for 1-5 wk, extended (Steridyl CSS One Step ®), and divided into 5 aliquots: control (no additive) or 0.5- or 1-mM of melatonin or L-arginine. For total and progressive motility (CASA), there were effects of group (P = 0.023 and P = 0.008, respectively); for morphological abnormalities (eosin-nigrosin), effects of group (P = 0.01) and a group*week interaction (P = 0.03); and for acrosome integrity (FITC-PSA), effects of group (P = 0.046) and week (P = 0.001). All 4 treatments improved motility (~5-10% points), whereas 1 mM of either compound optimized abnormalities and acrosomal integrity (~7% and 12% points, respectively). For superoxide dismutase and catalase, there were effects of week (P = 0.01 and P = 0.045, respectively), with 1 mM of either additive yielding best results. For DNA fragmentation index (DFI%), there was an effect of week (P = 0.01), and a group*week interaction (P = 0.05), with all 4 treatments reducing DFI%. For total ROS%, there was an effect of week (P = 0.044) and a group*week interaction (P = 0.037), with 1 mM melatonin or L-arginine being best. The hypothesis that melatonin or L-arginine improve quality of frozen-thawed sperm from HS rams was supported; 1 mM of either gave best results, except 0.5 mM minimized DFI%.

Scrotal subcutaneous temperature is increased by scrotal insulation or whole-body heating, but not by scrotal neck insulation; however, all three heat-stress models decrease sperm quality in bulls and rams.

Ruminant testes are ~2-6 °C below body temperature; increased testicular temperature reduces sperm motility and morphology. Our objective was to serially monitor scrotal subcutaneous temperatures during testicular heat stress and relate those to sperm quality. Two experiments were conducted, with temperature sensors surgically implanted in scrotal subcutaneous tissues recording temperatures every 15 min and semen collected and evaluated weekly. After an initial control interval, testicular temperature was increased. In Experiment 1, in two Angus bulls, whole-scrotum insulation for 96 h increased scrotal subcutaneous temperatures by ~2.0-2.5 °C (P < 0.05). Total and progressive motility decreased (P < 0.05) and reached a nadir at Week 3 (~20 and 10%, respectively). Furthermore, morphologically normal sperm and acrosome integrity also decreased significantly, reaching nadirs at Weeks 3 (15%) and 4 (34%). In Experiment 2, 10 Dorset rams were allocated randomly into two equal groups and either: 1) exposed to 28 °C ambient temperature and 30-34% RH for 8 h/d for 4 d; or 2) subjected to scrotal neck insulation that was applied and removed at the same time as the start and completion, respectively, of heat exposures in the other rams. Scrotal subcutaneous temperatures (monitored in three rams per group) were increased in response to whole-body heating (~0.8 °C, P < 0.05), but not significantly changed by scrotal neck insulation. Decreases in sperm quality were generally similar between treatments, with the most profound changes evident 4 wk after heat stress, with ~10 percentage point reductions in both total and progressive motility and ~10 and 20 percentage point decreases in morphologically normal sperm in rams with whole-body heating versus scrotal neck insulation, respectively. In conclusion, scrotal subcutaneous temperature was significantly increased by scrotal insulation or whole-body heating, but not by scrotal neck insulation; however, all three heat-stress models decreased sperm motility and morphology in bulls and rams.

Impact of supplementation of semen extender with antioxidants on the quality of chilled or cryopreserved Arabian stallion spermatozoa.

The aim of the present study was to evaluate the effects of supplementation of semen extender with various non-enzymatic antioxidants on the quality of cooled or cryopreserved Arabian stallion spermatozoa. Semen collected from four pure Arabian stallions was centrifuged at 600g for 15 min. Spermatozoa were then diluted in INRA-82 extender supplemented with bovine serum albumin (BSA; 0, 10, 15 and 20 mg/mL) or trehalose (0, 75, 100 and 150 mM) or zinc sulphate (0, 100, 150 and 200 µM). The diluted semen was then either cooled at 5 °C or cryopreserved in 0.5-ml plastic straws. After cooling or thawing, sperm motility, viability, sperm abnormalities, viability index, and plasma membrane integrity were evaluated. The results showed that supplementation of semen extender with 150 mM trehalose or with 200 µM zinc sulphate significantly (P < 0.05) improved motility, viability, sperm membrane integrity and acrosome status in Arabian stallion spermatozoa after cooling or after freezing and thawing compared with controls (non-supplemented media) or with those supplemented with other concentrations of trehalose or zinc sulphate. Supplementation of semen extender with BSA did not improve sperm motility or cryosurvival of Arabian stallion spermatozoa after cooling or after freezing and thawing. In conclusion, supplementation of semen extender with non-enzymatic antioxidants (trehalose or zinc sulphate) improved the quality of chilled and frozen/thawed Arabian stallion spermatozoa. The most beneficial effects occur when semen diluent was supplemented with 150 mM trehalose or 200 µM zinc sulphate.