Variation among stallions in sperm quality after single layer centrifugation

Although single layer centrifugation (SLC) selects robust spermatozoa from stallion semen, the effect of individual variation has not been studied in detail. The objective of this study was to determine the variation among stallions in the effects of SLC on sperm quality during cooled storage for up to 48 hr. Semen samples from seven stallions (18 ejaculates) were split, with one portion being used for SLC and the other serving as a control (CON). Sperm quality (kinematics, reactive oxygen species (ROS) production, membrane integrity (MI) and chromatin integrity) were analysed at 0, 24 and 48 hr using computer-assisted sperm analysis and flow cytometry. Sperm quality was better in SLC than in CON at all timepoints, especially chromatin integrity and MI (p < .0001 for both), and some categories of ROS production (e.g. proportion of live hydrogen peroxide negative spermatozoa, p < .0001), but the degree of improvement varied among stallions and type of ROS (p < .05–p < .0001). Total and progressive motility were also better in SLC samples than in CON at 24 and 48 hr (p < .0001), although the effect on sperm kinematics varied. The interaction of treatment, time and stallion was not significant. In conclusion, sperm quality was better in SLC samples than in CON, although there was considerable individual variation among stallions. The improvement in sperm quality, particularly in chromatin integrity, was clearly beneficial, and therefore the use of this technique would be warranted for all stallion semen samples.     

Supplementing a skimmed milk–egg yolk-based extender with L-carnitine helps maintain the motility,membrane integrity and fertilizing capacity of chilled ram sperm

This study examines the effect of L-carnitine (LC) on chilled ram semen stored for up to 96 hr. Semen samples were collected, placed in a skimmed milk + 6% egg yolk extender, pooled, aliquoted and diluted with the same extender supplemented with different LC concentration: 0 (control), 1 mM (LC1), 2.5 mM (LC2.5), 5 mM (LC5), 7.5 mM (LC7.5) or10 mM (LC10). Sperm kinetics and membranes (plasma, acrosome and mitochondrial) were examined using the CASA system and triple fluorescence staining (PI/ PNA-FITC/Mitotracker). The progressive motility was greater (p < .05) with LC7.5 treatment than the control sperm at 96 hr. The curvilinear velocity (p < .01) and the percentage of sperm with intact membranes (plasma/acrosome/mitochondria) (p < .01) were greater with all LC treatments than the control group at all times. Straight line velocity was greater (p < .01) with LC5 and LC7.5 treatments than the control group after 48 hr. The LC5 group also returned lower ALH values (p < .05) than these seen for the control groups after 48 hr. The fertilizing capacity of LC5 samples stored at 15°C for 2 hr (LC5-15°C-2h) and at 5°C for 24 hr (LC5-5°C-24h) was tested in three ewe groups via cervical fixed-time artificial insemination. In two groups, the fertilizing capacity of the LC5-5°C-24h was reduced (p < .001). In the remaining group, however, no significant difference was seen between the LC5-15°C-2h and LC5-5°C-24h sperm in this respect (pregnancy rates 52.4% versus 42.8%; p > .05). Overall, the present results suggest that supplementing skimmed milk–egg yolk-based extenders with LC has a positive effect on chilled sperm variables and fertilizing capacity.     

Semen quality improvement in boars fed with supplemental wolfberry (Lycium barbarum)

Wolfberry is well known for its health benefits in Asian countries. This study consisted of two experiments. In Experiment 1, nine boars were provided 40 g dried wolfberry per 100 kg body weight per day in addition to regular feed for 160 days (divided into 40 days phases: I, II, III, and IV) under step‐down air temperature conditions. Controls (n = 9) were fed regular feed only. Significant (p < .05 or p < .01) or slight improvements in sperm progressive motility, total abnormality rate, sperm concentration, and total sperm per ejaculate were observed in the wolfberry group during phases II and III. No differences were observed in semen volume. After combining the data from phases II ~ IV, significant improvements were detected in all aforementioned traits (p < .05 or p < .01), except semen volume. In Experiment 2, the wolfberry group (n = 5) was fed wolfberry for 90 days and exhibited significantly reduced head, tail, and total abnormality rates (p < .05 or p < .01) in both fresh semen and semen stored for 72 hr at 17°C compared to the control group (n = 5). SOD activity also significantly increased in this group of boars. Collectively, the findings of this study suggest that wolfberry has a positive effect on boar semen quality.     

Supplemented stallion seminal plasma can improve impaired motility due to the dilution effect in chilled Asian elephant sperm

The dilution effect and effect of restoring seminal plasma (SP) proportion in diluted semen were determined in chilled Asian elephant sperm. Semen was collected from eight males, and samples with ≥30% motile sperm were used in the study. Tris‐glucose‐egg yolk extender (TE) was used for cooled storage at 4°C for 48 hr. In experiment 1 (n = 18), semen was diluted to 1:1, 1:3, 1:7 and 1:15 with TE (volume per volume). There were no significant changes in sperm viability and sperm with normal acrosome integrity among dilutions, but sperm motility and motility velocities were greater (p < .05) in the 1:1 dilution than those of the 1:7 and 1:15 dilutions at 48 hr of storage. In experiment 2, supplemented SP was derived from elephants and stallions. In experiment 2.1, diluted semen (1:7 dilution) was restored with SP to obtain a 1:2 proportion (n = 8). Sperm motility, viability and sperm with normal acrosome integrity were similar among treatments, but motility velocities were greater (p < .05) with stallion SP at 48 hr of storage. In experiment 2.2, diluted semen (1:15 dilution) was restored with SP to obtain a 1:3 proportion (n = 10). Sperm viability and sperm with normal acrosome integrity were similar among treatments at 48 hr of storage. However, sperm motility and motility velocities were greater (p < .05) with stallion SP than those of others. In conclusion, elephant sperm motility was affected by a dilution effect and restoration of SP proportion with stallion SP, but not with elephant SP, could improve motility in chilled highly diluted sperm.     

Effect of feeding pomegranate seed oil as a source of conjugated linolenic acid on Arabian stallion semen quality in cooled and postthawed condition

The objective was to assess the influence of pomegranate seed oil supplementation on the quality of fresh, cooled and frozen–thawed Arabian breed stallion semen. Eight stallions (n = 4 per group) received their normal diet (control group) or normal diet top dressed with 200 ml of pomegranate seed oil (PSO group). Semen was collected every fifteen days for 90 days. Stallions were reversed across the treatments after a sixty‐day interval. In cooled and stored condition (2, 12 and 24 hr), spermatozoa motion characteristics, membrane integrity, viability, morphology and lipid peroxidation were analysed. In frozen–thawed semen, sperm dynamic characteristics were analysed by CASA, acrosome status and mitochondrial activity (evaluated by Flow cytometry) determined. The effects of treatment, time, semen type and their interactions were submitted to PROCMIX (SAS^®), and means compared by the Tukey test. Also, collected semen samples were artificially inseminated to evaluate fertility and pregnancy rate after day 60 of the experiment. The results from fresh condition showed that semen volume, sperm concentration, abnormality and live sperm were not affected by dietary treatment (p > 0.05). In cooled condition, the higher value for sperm plasma membrane integrity and viability was observed in PSO group compared to control after 24 hr cooled and stored in 5°C. In postthawed condition, the higher value for CASA total motility and acrosome status was observed in PSO group compared to control group (p < 0.05). One hundred and twenty‐six mares were artificially inseminated for fertility trial using control and PSO groups’ fresh semen. The average pregnancy rates were not significantly different between control and treated group (62.88% and 65.90%, respectively) (p > 0.05). We concluded that under the conditions of this study, dietary supplementation of 200 ml pomegranate seed oil seems to relatively improved Arabian horse sperm quality during storage in cooled and frozen condition via increasing plasma membrane integrity, viability and acrosome status, but did not improve the pregnancy rates.     

Epigallocatechin‐3‐gallate (EGCG) and green tea polyphenols do not improve stallion semen parameters during cooling at 4°C

Stallion semen storage for artificial insemination is mainly based on liquid cooled storage. In many stallions this technique maintains sperm quality for an extended period of time (24–72 hr) at 7°C. While this technique is commonly used in the horse industry, there can be a decline in fertility in some stallions, due to an inability of their sperm to tolerate the cool storage process. The aim of the present work was to evaluate the effect of two natural antioxidants (epigallocatechin‐3‐gallate (EGCG) at 20, 60 and 120 μm and green tea polyphenols, and p at .001, .01 and .1 mg/ml) on some sperm parameters (sperm motility, viability/acrosome integrity and DNA quality) in extended semen immediately after its collection (T0) and after 2, 6, 24 and 48 hr of cool storage. Two ejaculates from three trotter stallions were analysed after 48 hr of storage at 4°C. No beneficial effect on the analysed parameters was observed: the two antioxidants were not able to improve sperm quality after 48 hr of storage. These results are in agreement with previous findings on the effect of different antioxidants reported by other researches, who have demonstrated that stallion semen keeps good antioxidant capacity after dilution for 24 hr. In conclusion, the positive effect exerted by antioxidant molecules in other species is not confirmed in the equine one.     

Effect of semen extenders and storage time on quality of Muscovy duck (Cairina moschata) drake semen during the entire reproductive season

Avian semen dilution with appropriate extender allows to prolong the fertilizing ability of sperm stored in vitro. In the present study, the impact of extenders and time of storage on morphology of Muscovy duck (Cairina moschata) drake semen were examined. Semen was collected twice a week, using male stimulation by a female method, from 12 adults (29 weeks old) drakes kept individually in cages, under controlled environmental conditions. Freshly collected, pooled ejaculates were divided into three part: neat undiluted sample, and diluted 1:1 with Schramm (SCH) or Watanabe (W) extender and stored at 4°C. Morphological examination of all samples was conducted after dilution and then, after 3 and 6 hr of storage. The storage of undiluted semen caused decrease (p ≤ .01) in live morphologically normal sperm, from 79.73% in the freshly collected ejaculates to 55.75% and to 12.12% after 3 and 6 hr of storage, respectively (average calculated for the entire reproductive season). In the semen diluted with Schramm's extender the adequate values attained 86.84, 79.65 and 61.66%, and using Watanabe extender 84.77, 83.58 and 75.25%, respectively. The period of semen storage and the type of extender caused significant (p ≤ 0,05; p ≤ 0,01) changes in sperm morphology. The longer period of storage contributed to the decrease in number of morphologically normal sperm, whereas their content in Watanabe extender after 3 and 6 hr of storage was higher (p ≤ .01) than in semen diluted in Schramm extender.     

Performance,body fat reserves and plasma metabolites in Brown Swiss dairy cows: Indoor feeding versus pasture‐based feeding

Feeding dairy cows indoors or on pasture affects not only labour, machinery and housing costs, but also animals’ performance and metabolism. This study investigates the effects of indoor feeding (IF) with a partial‐mixed ration (PMR) versus pasture‐based feeding (PF) on milk production, fertility, backfat thickness (BFT), body weight (BW) loss and energy metabolism of Brown Swiss (BS) dairy cows with similar genetic production potential. The IF herd consisted of 13 cows fed a PMR composed of maize and grass silage plus protein concentrate according to each cow's requirements. The PF herd consisted of 14 cows offered barn‐ventilated hay ad libitum after calving from January until March and grazed on semi‐continuous pastures during the vegetation period. The IF cows produced more energy‐corrected milk (ECM) per standard lactation (9,407 vs. 5,960 kg; p < .01), more milk fat (378 vs. 227 kg; p < .01) and milk protein (326 vs. 215 kg; p < .01). The calving interval (377 vs. 405 days; p < .01) and time empty (86 vs. 118 days; p < .01) were shorter in the PF compared to IF, possibly also due to different selection criteria for maintaining the respective seasonal calving rhythm. The empty body fat loss calculated according to BCS until its nadir was higher in IF cows (IF: 10.4 vs. PF: 4.8 MJ/day; p < .01), but no differences were noted in total body fat loss estimated via BFT (p = .24). However, PF had lower blood glucose concentration at all investigated time points, but no differences occurred in serum non‐esterified fatty acid and β‐hydroxybutyrate concentrations post‐partum. In conclusion, BS cows were equally well suited for the IF with PMR and the PF system investigated here without developing a prominent metabolic load despite differences in nutrient supply. As such, investigated BS dairy cows in our trial seem to have a high capacity for metabolic adaptation to different production systems.     

In vitro studies of Norwegian Red bovine semen immobilized and cryopreserved in alginate solid gel network

Development of new semen cryopreservation techniques improving sperm survival and ensuring availability of viable spermatozoa for a prolonged time‐period after AI is promising tools to reduce sensitivity of timing of AI and enhance overall fertility. The SpermVital^® technology utilizes immobilization of bull spermatozoa in a solid network of alginate gel prior to freezing, which will provide a gradual release of spermatozoa after AI. The objective of this study was to compare post‐thaw sperm quality and in vitro sperm survival over time of Norwegian Red bull semen processed by the SpermVital^® (SV) technology, the first commercialized production line of SpermVital^® (C) and by conventional procedure applying Biladyl^® extender (B). Post‐thaw sperm motility was not significantly different between SV, C and B semen (p > .05). However, sperm viability and acrosome intactness were higher for SV than C and B semen (p < .05). Small differences in DNA quality were observed (p < .05). Sperm viability after storage in uterus ex vivo was higher for SV than for C semen (p < .05). Furthermore, sperm survival in vitro over time at physiological temperature was significantly higher for SV semen than C semen as well as B semen during the incubation period of 48 hr (p < .05). In conclusion, the SpermVital^® technology is improved and is more efficient in conserving post‐thaw sperm quality and results in higher sperm viability over time in vitro for SV than for C and B semen.     

Deoxynivalenol induces oxidative stress,inflammatory response and apoptosis in bovine mammary epithelial cells

Deoxynivalenol (DON) is a toxic secondary metabolite produced by Fusarium graminearum. It is one of the most common feed contaminants that poses a serious threat to the health and performance of dairy cows. This study investigated the in vitro cytotoxicity of DON on bovine mammary epithelial cells (MAC‐T). DON at different concentrations (0.25, 0.3, 0.5, 0.8, 1 or 2 μg/ml) inhibited the growth of MAC‐T cells after 24 hr of exposure (p < .001). DON at 0.25 μg/ml increased lactate dehydrogenase (LDH) leakage (p < .05); decreased glutathione (GSH) levels (p < .001), total superoxide dismutase (T‐SOD) activity and total antioxidant capacity (T‐AOC; p < .01); and increased malondialdehyde (MDA) concentration (p < .01) in MAC‐T cells after 24 hr of exposure. We also observed that DON increased reactive oxygen species (ROS) levels in cells incubated for 9, 15 and 24 hr (p < .001). DON at 0.25 μg/ml triggered oxidative damage in MAC‐T cells. Furthermore, it induced an inflammatory response in the cells incubated for 9, 15 and 24 hr (p < .05) by increasing the mRNA expression levels of nuclear factor kappa B, myeloid differentiation factor 88 (MyD88), tumour necrosis factor‐α (TNF‐α), interleukin‐1β (IL‐1β), IL‐6, cyclooxygenase‐2 and IL‐8. We further examined the effect of DON on apoptosis. DON prevented normal proliferation of MAC‐T cells by blocked cell cycle progression in 24 hr (p < .001). In addition, the apoptosis rate measured using annexin V‐FITC significantly increased (p < .05) with increase in the mRNA expression level of Bax (p < .01) and increase in the Bax/Bcl‐2 ratio (p < .01) in cells incubated for 24 hr. In summary, DON exerts toxic effects in MAC‐T cells by causing oxidative stress, inducing an inflammatory response, affecting cell cycle and leading to apoptosis.     

Investigation of in vitro measurable sperm attributes and their influence on electroejaculated bull semen with a fixed‐time artificial insemination protocol in Australian Bos indicus cattle

Increasing use of fixed‐time artificial insemination (FTAI) in beef cattle production has presented an opportunity for the use of fresh or chilled semen as an alternative to standard cryopreserved semen. The objective of this study was to examine in vitro sperm function and pregnancy rate of electroejaculated semen, chilled and stored for 48 hr, compared to conventionally cryopreserved semen with an optimized FTAI protocol in Brahman cattle. Semen from three Brahman bulls was collected, and aliquots were extended in either chilled (at 5°C) or frozen (LN₂) in a Tris‐egg yolk extender base with 2.4% or 7.0% glycerol, respectively. Semen samples were assessed 48 hr after collection or post‐thaw and warming, for sperm motility, in vitro sperm function and fertilizing ability, and used in a FTAI programme. The overall pregnancy rates was significantly different (p < .01) after FTAI with frozen (n = 173; 53.2%) and chilled semen (n = 174; 31.6%). In contrast, the in vitro sperm assessment showed that the chilled semen had significantly faster motility (p < .05), a higher proportion of progressively motile spermatozoa (p < .05), with significantly higher proportions of acrosome intact, viable spermatozoa (p < .01). This study showed that reasonable pregnancy rates in Brahman cattle can be achieved using FTAI with chilled semen collected using electroejaculation and stored for up to 48 hr. However, improvements in semen extenders are required in consideration of semen collection method to improve the longevity of sperm fertilizing ability to significantly increase FTAI output using chilled storage of bull semen.     

Antioxidants protect proteins' anchorage to the bilayer by improving plasma membrane integrity of ram spermatozoa during liquid preservation in a soya lecithin‐based diluent

Antioxidants are known to prevent the reactive oxygen species (ROS)‐mediated peroxidative damage to the membrane lipids during hypothermic storage of mammalian spermatozoa. We hypothesized here that ROS also affect the lipid–protein interactions, thereby diminishing the membrane's integrity and proteins' anchorage to the bilayer. Antioxidants prevent these damages by scavenging the ROS. Ejaculates from Patanwadi rams were pooled after subjective evaluation and centrifuged using Percoll^®. Sperm pellet was resuspended in soya lecithin–Tris–fructose diluent (400 × 10⁶ cells/ml) containing either antioxidants (100 IU/ml catalase + 10 mM reduced glutathione) or no antioxidant. Aliquots were chilled to 5°C in a cabinet and stored in a refrigerator at 3–5°C for 72 hr. Sperm motility, viability, lipid peroxidation (LPO) and hypo‐osmotic swelling test (HOST) were performed at 0, 24, 48 and 72 hr. Sperm proteins extracted with 0.5% Triton X‐100 were resolved by SDS‐PAGE and quantified using Quantity One software (Bio‐Rad, USA). The rapid motility, linearity and straight‐line velocity (VSL) were found significantly (p < .05) higher in the antioxidant‐treated group compared to the control at 48 hr of storage. Sperm viability was found comparable between the groups. Higher HOST response and lower LPO were found in the antioxidant‐treated sample compared to the control both at 48 and at 72 hr. Overall, the proteins P1 (106.09 kDa), P2 (87.00 kDa) and P4 (51.14 kDa) were lower (p < .05) in the sperm extract of antioxidant‐treated group compared to the control. The content of P4 (51.14 kDa) in sperm extract was found to increase (p < .05) earlier (48 vs. 72 hr) in the control group compared to the antioxidant‐treated group. Altogether, the results suggested that antioxidants reduced LPO in spermatozoa, resulting in higher sperm motility, plasma membrane integrity and protection of proteins' anchorage to the plasma membrane at 48 and 72 hr of storage.     

Semen supplementation with palmitoleic acid promotes kinematics,microscopic and antioxidative parameters of ram spermatozoa during liquid storage

The purpose of the present experiment was to investigate the protective effects of palmitoleate on the quality of ram semen during low temperature liquid storage. Ejaculates were collected using the artificial vagina from four Qezel rams twice a week. Ejaculates were pooled, diluted with Tris–egg yolk extender without palmitoleate (control) or supplemented with 0.125 (P 0.125), 0.25 (P 0.25), 0.5 (P 0.5) and 1 (P 1) mM palmitoleate at a final concentration of 500 × 10⁶ spermatozoa/ml. Total motility and forward progressive motility (FPM) as well as other spermatozoa kinematics were evaluated by computer‐assisted sperm analysis. Moreover, viability and membrane functionality were determined in the spermatozoa. Additionally, amounts of malondialdehyde (MDA), total antioxidant activity (AOA), nitric oxide (NO) and superoxide dismutase (SOD) activities were evaluated in the medium and spermatozoa at 0, 24, 48 and 72 hr of storage. The palmitoleate supplementation resulted in a significant (p < .05) increase in total motility and FPM with the highest increase at 0.5 mM concentration for 72 hr. P 0.5 group also resulted in the highest percentage of membrane‐intact spermatozoa (76.60 ± 1.95%) and viability (75.81 ± 1.34%) at 72 hr (p < .05). The amounts of MDA and NO were lower in P 0.125, P 0.25 and P 0.5 groups compared to control at 48 hr and 72 hr (p < .05). Higher amounts of AOA were obtained in palmitoleate‐treated groups in medium and spermatozoa during storage time (p < .05). Furthermore, palmitoleate supplementation increased the SOD activities in spermatozoa compared to the control (p < .05). The results of the present experiment reveal that supplementation with 0.5 mM palmitoleate improves ram spermatozoa motion characteristics, AOA levels and SOD activities during liquid storage. Then, palmitoleate could be used as an antioxidant source during liquid storage of ram semen.     

Orally administered Chrysin improves post‐thawed sperm quality and fertility of rooster

Chrysin is a bioflavonoid compound found in passion flower, chamomile, propolis and honey at high levels. Post‐thawed sperm quality and fertility of Chrysin‐fed roosters were assessed in this study. Twenty 40‐week‐old male broiler breeders were randomly divided into four groups and fed basal diet supplemented with different levels of Chrysin including 0 (Ch‐0), 25 (Ch‐25), 50 (Ch‐50) or 75 (Ch‐75) mg/day for 12 consecutive weeks. Semen samples were weekly collected from 6th to 9th week of experiment to evaluate some sperm quality parameters including total and progressive motility, plasma membrane integrity and functionality (in fresh and post‐thawed samples) and mitochondrial activity (only in post‐thawed samples). Also, collected semen samples from 10th, 11th and 12th week of experiment were frozen and then artificially inseminated to test fertility rate. According to the results, an improvement in both fresh and post‐thawed sperm quality including total [fresh: 88.00 ± 0.58 and 87.25 ± 0.67 (p < .01); post‐thawed: 51.07 ± 2.05 and 52.72 ± 1.96 (p < .01)] and progressive motility [fresh: 76.00 ± 0.58 and 78.25 ± 0.65 (p < .01); post‐thawed: 40.61 ± 2.01 and 39.88 ± 2.01 (p < .01)], plasma membrane integrity [fresh: 91.60 ± 0.58 and 89.85 ± 0.59 (p < .01); post‐thawed: 56.99 ± 1.86 and 54.39 ± 1.86 (p < .01)] and functionality [fresh: 75.40 ± 0.42 and 77.90 ± 0.96 (p < .01); post‐thawed: 45.69 ± 1.71 and 46.35 ± 1.71 (p < .01)] was noted for both Ch‐50 and Ch‐75, respectively, groups compared to control group. Despite no significant change in mitochondrial activity, fertility rate of post‐thawed spermatozoa was significantly improved in all Chrysin‐fed groups compared to Ch‐0 group. In conclusion, oral Chrysin administration to roosters could ameliorate cryopreservation‐induced impairment of sperm quality and fertility rate.     

Transport stress induces pig jejunum tissue oxidative damage and results in autophagy/mitophagy activation

Pig transportation is associated with intestinal oxidative stress and results in destruction of intestinal integrity. Autophagy has been contributed to maintain cell homeostasis under stresses. The purpose of this study was to evaluate the effects of transport stress on morphology, intestinal mucosal barrier and autophagy/mitophagy levels in pig jejunum. A total of 16 finishing pigs were randomly divided into two groups. The control group was directly transported to the slaughterhouse and rested for 24 hr. The experimental groups were transported for 5 hr and slaughtered immediately. The results showed that transportation induced obvious stress responses with morphological and histological damage in jejunum accompanying with an elevated level of malondialdehyde (MDA; p < .05), endotoxin (LPS; p < .05), lactic dehydrogenase (LDH; p < .05) and a decreased level of serum superoxide dismutase (SOD; p < .05). Also, hemeoxy genase 1 (HO‐1; p < .01) as well as tight junction protein (claudin‐1 [p < .001], occludin [p < .05] and zonula occludens 1 [ZO‐1; p < 0.05]) levels were attenuated in jejunum tissue, and NADPH oxidase 1 (NOX1; p < .01) mRNA expression was up‐regulated. Further research indicated that transport stress could induce autophagy through increasing microtubule‐associated protein light chain 3 (LC3; p < .05) and autophagy‐related gene 5 (ATG5; p < .01) levels and suppressing p62 expression. Additionally, transport stress increased the protein levels of PTEN‐induced putative kinase 1 (PINK1; p < .05) and Parkin (p < .05) which was associated with mitophagy. In conclusions, transport stress could induce the destruction of intestinal integrity and involve in the intestinal mucosal barrier oxidative damage, and also contribute to activation of autophagy/mitophagy.     

Does single-layer centrifugation with Bovicoll improve sperm quality of frozen-thawed semen in Fleckvieh bulls?

The aim of the present study was to evaluate the effect of sperm selection by single-layer centrifugation (SLC) performed before freezing on sperm quality after thawing of Fleckvieh bull semen. Ejaculates from 22 bulls were collected by artificial vagina and divided into two aliquots. One aliquot (control sample) was diluted with Steridyl^® and frozen over nitrogen vapour in a Digitcool freezer (IMV Technologies). Sperm from the second aliquot (SLC sample) was selected using the SLC technique with Bovicoll colloid and then frozen over nitrogen vapour in a Digitcool freezer. After thawing, both samples (control and SLC) were evaluated by computer-aided sperm analysis (CASA; SCA 6.4 System; Microptic S.L) for sperm motility parameters. Integrity of the plasma membrane (viability), high mitochondrial membrane potential (HMMP) and acrosome integrity were assessed using a Guava® easyCyte flow cytometer (IMV Technologies). Morphological examination of spermatozoa was performed by Differential Interference Contrast microscopy (Leica DMi8). Morphological examination of live, immobilized spermatozoa was analysed under high magnification (≥6,600×). After thawing, the mean sperm viability of the control sample was 51.57%, compared to 40.37% for the SLC sample (p < .01). HMMP was higher (p < .01) in the control sample (40.37% versus 28.96%), and the mean of live spermatozoa with damaged acrosome was significantly higher (p < .03) in the SLC sample (1.63% versus 1.95%). The mean percentage of motile spermatozoa was 80.17% in the control sample, compared to 75.14% in the SLC sample (p < .0195), and rapid subpopulation reduced from 20.08% to 8.99% (p < .0001) after SLC. Percentage of hyperactivated sperm decreased from 12.23% to 4.28% (p < .0001) after SLC. Given the overall results, the sperm quality of thawed Fleckvieh bull semen was not improved when sperm were selected by SLC before freezing.     

Cooled Storage of Canine Semen: in vitro Effects of Different Concentrations of an Antibiotic Combination on Growth of Mollicutes

The aim of this study was to examine effects of an antibiotic combination at different concentrations on growth of mycoplasma and ureaplasma during cooled storage of canine semen (n = 20). Semen aliquots were diluted with Tris–citric acid–fructose–egg yolk extender containing either 1.0 g/l streptomycin and 0.6 g/l benzylpenicillin (control) or a combination of gentamycin, tylosin, lincomycin and spectinomycin (GTLS‐1: 0.25, 0.05, 0.15 and 0.3; GTLS‐2: 0.5, 0.1, 0.3 and 0.6; GTLS‐3: 1.0, 0.2, 0.6 and 1.2 g/l). Samples were assessed for motility and membrane integrity by computer‐assisted sperm analysis immediately after dilution and at 24, 48 and 72 h of cooled storage. Morphologically, normal spermatozoa were determined, and bacterial culture was performed at 24 and 72 h. Mycoplasma spp. were detected in 14 of 20 ejaculates (70%) with severe growth in 12 samples. A reduction but not total elimination of mycoplasma growth occurred in all GTLS extenders with the most pronounced reduction in group GTLS‐3 (control vs GTLS‐1 and GTLS‐2 p < 0.05, control vs GTLS‐3 p < 0.001). Ureaplasmas were detected in four ejaculates, and growth was reduced to the same extent in GTLS and control extender. Progressive motility in all groups, total motility in groups GTLS 1–3 and percentage of membrane‐intact spermatozoa in groups GTLS 2 and 3 decreased slightly (p < 0.05) over time. In conclusion, dilution of canine semen with GTLS extender has no major detrimental effects on spermatozoa during cooled storage. It reduced the growth but did not totally eliminate mycoplasmas and ureaplasmas from cooled‐stored dog semen.     

Influence of idebenone on ram semen quality stored at 4°C

The current study was designed to investigate the effect of idebenone (Id), an antioxidant on ram semen quality. Semen samples were collected, pooled and diluted in a Tris‐based extender supplemented with 0, 1, 2, 4 and 8 µM idebenone. Computer‐assisted sperm analysis was used to evaluate spermatozoa kinematics. Sperm viability and membrane functionality were assessed respectively, by eosin‐nigrosin staining and HOS test. Biochemical assays were carried out to measure different metabolites in spermatozoa and medium at 0, 24, 48 and 72 hr. Total and forward progressive motility were greater in 1, 2 and 4 µM idebenone treated groups compared to control at 24, 48 and 72 hr time points (p < 0.05). Semen supplementation with Id significantly increased viability and functionality of spermatozoa membrane during storage (p < 0.05). Lower amounts of lipid hydroperoxides in medium and spermatozoa were observed in Id‐treated groups compared to control one at 24 and 48 hr of storage (p < 0.05). Medium and spermatozoa amounts of malondialdehyde and nitric oxide were less in Id 4 µM group compared to the control at 72 hr (p < 0.05). Total antioxidant capacity values and superoxide dismutase activity of spermatozoa and medium were greater in 2 and 4 µM idebenone treated groups in comparison with the control at 72 hr (p < 0.05). Results of the current study indicated that ram semen supplementation with Id at 4 µM level improved quality by ameliorating nitrosative and peroxidative stress, hence could be considered as an antioxidant additive during storage at 4°C.     

Reproductive performance of immobilized cryopreserved bovine semen used for timed artificial insemination

The SpermVital^® technology comprises embedding of spermatozoa within an alginate gel to facilitate release of sperm cells over a prolonged period in utero after AI. The aim of this study was to examine whether the survival time of spermatozoa is extended when applying this immobilization technology in combination with cryopreservation. Sperm cell survival (acrosome and plasma membrane integrity) was studied in vitro for 48 hr at physiological temperature. One dose of SpermVital^® (SV) semen was compared with single doses of Biladyl^® (B) processed semen as well as double doses of B (B double). B double was obtained by adding a second B dose the following day, thereby mimicking double AI. Furthermore, reproductive performance applying single early timed AI (TAI) with SV following oestrus synchronization was studied in a field trial. Double insemination (TAI on two consecutive days) with B semen served as control. Number of acrosome‐intact live sperm cells decreased over time in vitro for all treatments (p < .05). There was no difference between SV sperm cell survival and B double after 24 hr (p > .05). However, after 48 hr, SV sperm cell survival was higher than B double (p < .05). Moreover, multivariate analysis showed that the outcome of single early TAI with SV was not significantly different from B double (p > .05). Likelihood of pregnancy and calving in the heifer group was higher than in the cow group (p < .05). These results imply that spermatozoa immobilized in alginate gel have prolonged survival.     

Combined cryopreservation of canine ejaculates collected at a one-hour interval increases semen doses for artificial insemination without negative effects on post-thaw sperm characteristics

A limiting factor in canine artificial insemination (AI) is the low number of insemination doses obtained per ejaculate. In this study, semen was collected from dogs (n = 28) either once and frozen directly after collection or the same dogs were submitted to a dual semen collection with a 1-hr interval and the two ejaculates were combined for cryopreservation. We hypothesized that combining two ejaculates increases semen doses per cryopreservation process without negative effects on semen characteristics. Total sperm count was lower in semen from a single semen collection in comparison with the combination of the first and second ejaculate of a dual semen collection (p < .001). The percentage of motile and membrane-intact spermatozoa determined by computer-assisted sperm analysis (CASA) in raw semen did not differ between single and combined dual ejaculates and was reduced (p < .001) by cryopreservation to the same extent in single (motility 73.7 ± 1.8%, membrane integrity 65.6 ± 2.2%) and combined dual ejaculates (motility 72.7 ± 2.3%, membrane integrity 64.6 ± 2.5%). The percentage of spermatozoa with morphological defects increased after cryopreservation (p < .001) but was similar in single and combined dual ejaculates. The CASA sperm velocity parameters decreased with cryopreservation (p < .001) but did not differ between single and combined dual ejaculates. The number of insemination doses increased from 2.7 ± 0.4 for single to 4.7 ± 0.8 for combined dual ejaculates (p < .01), based on 100 million motile spermatozoa per frozen-thawed semen dose. In conclusion, combining two ejaculates collected at short interval for one cryopreservation process increases the number of AI doses without compromising semen quality.