A soy lecithin nanoparticles-based extender effectively cryopreserves Holstein bull sperm.

Plant-based semen extenders, typically derived from soybean lecithin, are easier to modulate more and consistent in their composition than animal-based extenders. As large lecithin particles can, however, reduce effectiveness and solubility in bull semen extenders, sonication was used to create nano-lecithin (NL) particles of soybean lecithin. The objective was to determine the effects of lecithin type and concentration on the quality of frozen-thawed bovine sperm. We hypothesized that reducing the size of lecithin improves its interactions with the sperm and enhances the parameters that favor its motility, viability and fertility. Semen was collected from six mature Holstein bulls and ejaculates meeting minimum standards were pooled. Eight Tris-based extenders that contained 1, 2, 3, or 4 % of either conventional lecithin (L1-L4) or NL (NL1-NL4), plus two control extenders (one animal-based extender containing 20 % egg yolk [EY] and a commercial lecithin-based extender [BioXcell®]) were compared. Among soybean lecithin-based extenders, NL3 had the highest total and progressive sperm motility, and average path, straight-line and curvilinear sperm velocity, and was comparable to EY. Additionally, sperm mitochondrial activity was the highest in NL3, whereas sperm viability was highest in EY, NL3, and L4. Following in vitro fertilization of in vitro-matured bovine oocyes, NL3 had cleavage and hatching rates comparable to BioXcell®, but a lower blastocyst rate than EY. Overall, NL3 performed better than the other extenders for most end points, with efficiency comparable to EY. We, therefore, concluded that reducing lecithin particle size to a nano level improves sperm cryopreservation with optimal performance with 3 % NL.

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.

Supplementation of freezing medium with encapsulated or free glutathione during cryopreservation of bull sperm.

The objective was to compare effects of encapsulated or free glutathione (GSH) on the quality of frozen-thawed bull sperm. Ejaculates were collected via artificial vagina from six mature Holstein bulls once weekly for 6 weeks. All ejaculates had motility ≥70%, sperm concentration ≥1.0 × 109 /ml and ≤15% morphologically abnormal sperm. Each week, semen was pooled and diluted with lecithin-based extenders containing various concentrations of encapsulated (E0, E1, E2.5 and E5 mM) or free (F0, F1, F2.5 and F5 mM) GSH, with total glutathione content determined before and after cryopreservation. Total GSH in fresh semen was (mean+SEM) 4.8 ± 0.2 nmol/108 sperm, whereas in frozen-thawed semen of group F0 (control), it decreased to 1.4 ± 0.2 nmol/108 sperm, a 70.8% reduction (p < .05). In addition, total GSH in frozen-thawed semen from groups E2.5, E5 and F5 were 2.4 ± 0.2, 2.8 ± 0.2 and 1.8 ± 0.2 nmol/108 sperm, respectively (E5 versus. F0, p < .05). Compared to group F0, frozen-thawed sperm from group E2.5 had greater (p < .05) percentages of sperm that were viable (Annexin-V) (61.1 ± 1.8 versus. 71.1 ± 1.8) and that had cell membrane integrity (eosin-nigrosin) (64.5 ± 3.1 versus. 80.0 ± 3.1). Furthermore, frozen-thawed sperm from group E2.5 had the numerically highest total and progressive motility (CASA) and cell membrane functionality (HOS) and the lowest percentage of early apoptotic sperm (Annexin-V). However, acrosome membrane integrity (PSA) of E5 had the lowest mean (p < .05), whereas E2.5 caused a small nonsignificant decrease (69.1 ± 1.4%) compared to E0 and F0. In conclusion, 2.5 mM encapsulated GSH in semen extender significantly improved the quality of frozen-thawed bull sperm.

Dietary omega-3 fatty acids from linseed oil improve quality of post-thaw but not fresh sperm in Holstein bulls.

Docosahexaenoic acid (DHA), a member of the n-3 fatty acid family present in fish oil, has several positive effects on bovine sperm, including membrane integrity, motility and viability, as well as cold sensitivity. Our objective was to investigate effects of varying amounts of omega-3 fatty acids from linseed oil, administered orally, on quality of fresh and frozen-thawed bull sperm. Twenty fertile Holstein bulls (874 ± 45.38 kg) were randomly and equally assigned to four groups and received encapsulated (rumen-protected) fats for 12 weeks, as follows: group P, 300 g palm oil; group Pl, 200 g palm oil + 100 g linseed oil; group pL, 100 g palm oil + 200 g linseed oil; and group L, 300 g linseed oil. Sperm quality of fresh and frozen-thawed semen was evaluated by routine assays including sperm motion characteristics (CASA), membrane integrity (eosin-nigrosin), membrane activity (hypo-osmotic swelling test; HOST) and malondialdehyde (MDA) content. There were no significant differences among groups in semen volume, sperm concentration or sperm quality parameters in fresh semen. However, after freezing-thawing, total and progressive motility in group P (59.61 ± 1.95 and 40.19 ± 2.48%, respectively; LSM ± SEM) were lower (P < 0.05) than in groups Pl (66.06 ± 1.95 and 47.53 ± 2.48%), pL (65.67 ± 1.95 and 47.48 ± 2.48%) and L (65.36 ± 1.95 and 47.62 ± 2.48)%, with no significant differences among the latter three groups. Furthermore, membrane integrity (eosin-nigrosin) and activity (HOST) were lower (P < 0.05) in group P (55.79 ± 2.15 and 42.19 ± 2.17%) compared to groups Pl (62.73 ± 2.15 and 48.93 ± 2.17%), pL (64.06 ± 2.15 and 50.01 ± 2.17%) and L (64.47 ± 2.15 and 49.68 ± 2.17%), with no significant differences among the latter three. Furthermore, there were more (P < 0.05) morphologically abnormal sperm in group P (25.99 ± 1.62%) than in groups Pl, PL and L (21.55 ± 1.62, 21.69 ± 1.62 and 20.90 ± 1.62%). In conclusion, feeding Holstein bulls 100-300 g linseed oil daily improved sperm cryotolerance.

Enhanced pre-pubertal nutrition upregulates mitochondrial function in testes and sperm of post-pubertal Holstein bulls.

Supplemental energy and protein during calf-hood (2-30 wk) in dairy bulls hastened puberty (~1 mo), upregulated steroid biosynthesis, concentrations of reproductive hormones and Sertoli cell maturation, with larger testes and greater sperm production (~25%) in mature bulls. The objective was to evaluate effects of feeding high (20.0% crude protein [CP], 67.9% total digestible nutrients [TDN]), control/medium (17.0% CP, 66.0% TDN) and low (12.2% CP, 62.9% TDN) diets from 2 to 30 wk on post-pubertal testes of Holstein bulls. Based on RNA sequencing, 497 and 2961 genes were differentially expressed (P < 0.1) in high- vs low- and high- vs medium-diet groups, respectively. According to KEGG analysis, oxidative phosphorylation and ribosome pathways were upregulated in high- vs medium- and low-diet groups, with majority of upregulated genes encoding for essential subunits of complex I, III, IV and V of OXYPHOS pathway. In addition, mitochondrial translation, mitotic nuclear division and cell division were enriched in high- vs medium-diet groups. Consistent with these results, a greater percentage of sperm from high-diet bulls were progressively motile and had normal mitochondrial function compared to medium-diet sperm (P < 0.1). Thus, enhanced early life nutrition upregulated mitochondrial function in testes and sperm of post-pubertal Holstein bulls.