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The decrease in fertility in aging roosters is related to the reduced quality of ejaculated sperm. This study aimed to investigate the effect of dietary supplementation with ginseng extract at various concentrations (0–150 mg/kg) on testicular function, semen preservation, and fertility at different stages of sexual maturity (mature and aging roosters) in Thai native roosters. Pradu Hang Dum roosters at 32 (mature; n = 24) and 75 (aging; n = 24) weeks of age were fed diets with non-supplemented or supplemented ginseng extracts (50, 100, and 150 mg/kg) until the end of the experiment. In experiment 1, fresh semen samples were examined for the quality parameters of semen volume, sperm concentration, sperm motility, sperm viability, lipid peroxidation, and enzymatic activities. In experiment 2, semen was preserved at 5 °C for up to 48 h, and the semen quality and fertility potential were determined. In experiment 3, testicular function and testosterone concentrations were evaluated. The results showed that ginseng extract supplementation in the diets of both mature and aging roosters at 50 and 100 mg/kg improved fresh semen quality (P < 0.05). A decrease in malondialdehyde levels in fresh semen was observed with increasing enzyme activities. In mature roosters, the progressive motility of cold-stored semen and fertility rates were higher in the G50 and G100 groups compared to the control and G150 groups after 24 h of storage (P < 0.05). In aging roosters, the highest significant differences in progressive motility, viability, and fertility rates were observed in the G50 and G100 groups at all storage times (P < 0.01). These improvements might be attributed to good testicular function in spermatogenesis, as revealed by the results of histological examination and testosterone concentrations. However, higher doses of ginseng extract supplementation negatively affected sperm quality. In summary, the recommended dose of ginseng extract supplementation in diets is 50 mg/kg. Fertility results indicated that insemination with semen preserved for 24 h was satisfactory in both mature and aging roosters. • Ginseng extract improved sperm production and semen preservation in mature and aging roosters. • Semen quality was highest in G50 and G100 groups, confirmed by histological and testosterone results. • High doses (150 mg/kg) negatively impacted sperm quality. • 24 hour preserved semen showed satisfactory fertility rates in both mature and aging roosters. [ABSTRACT FROM AUTHOR]

The aim of the study was to determine the effect of carbetocin administration (a long-acting analog of oxytocin) 20 or 10 min before electroejaculation (EE) on the duration of semen collection procedure, quantitative and qualitative characteristics of the ejaculate, and stress biomarkers in rams. Semen was collected from 12 Corriedale rams (age, 2.5–5.5 years old) with EE, in a Latin-square design, administrating carbetocin (0.2 mg/100 kg of body weight i.v.) 20 or 10 min before EE, or without carbetocin administration (CB-20, CB-10, and CON treatments, respectively). Each treatment was applied to different rams every 3–4 days, allowing all the rams to receive all three treatments. Carbetocin administered 20 or 10 min before EE increased the number of sperm ejaculated (P = 0.01), the semen concentration (P = 0.02), the number of insemination doses collected in a single collection (P = 0.01), and the number of insemination doses collected/electrical pulses administered (P = 0.05) compared to control rams. Carbetocin administered 20 or 10 min before semen collection prolonged the time required for EE and the number of pulses administered during EE compared to CON rams (P < 0.03 for both). The CB-10 rams required the administration of more electrical pulses during ejaculation than CON rams (P = 0.001), and CB-20 treatment tended to require more electrical pulses than CON rams (P = 0.06). The volume of the ejaculate was greater in CB-10 than in CON rams (P = 0.01), and that of CB-20 treatment tended to be greater than CON rams (P = 0.08). The percentage of sperm with intact membrane was greater in CB-20 than in CON rams (P = 0.01). Total protein, albumin, and globulin concentrations were lower immediately after carbetocin administration 20 or 10 min before EE. The treatments did not affect cortisol concentration, glycemia, rectal and surface temperatures, heart rate, and facial expressions. Carbetocin administration before EE of rams improved the quantitative and qualitative characteristics of the ejaculate, duplicating the number of insemination doses collected. It can be a promising treatment to obtain a greater quantity of doses to inseminate with a lower frequency of semen collections, reducing the negative impacts of EE on animal welfare. • Carbetocin administration before electroejaculation enhanced volume and sperm concentration in rams. • The number of insemination doses was doubled following carbetocin administration. • Percentage of membrane-intact sperm also increased with carbetocin. • Carbetocin use can be a complement in semen collections with electroejaculation. [ABSTRACT FROM AUTHOR]

The supplementation of sperm culture media with serum is quite common, and improves both sperm survival and motility. However, the link between serum and sperm remains poorly understood. The present study is the first investigation of the effects on sperm quality and function of endogenous porcine serum exosomes in medium used for culturing boar sperm. Scanning electron microscopy (SEM) confirmed that serum-derived exosomes from both castrated boars (cbsExos) and sows (ssExos) exhibited typical nanostructural morphology and expressed CD63, CD9, and Alix, as shown by Western blotting. At 17 °C, the progressive motility and membrane integrity of sperm were significantly increased after incubation of fresh boar semen for 7 days with cbsExos-4 (8 × 1010 particles/mL) or ssExos-16 (32 × 1010 particles/mL). Moreover, cbsExos-4 and ssExos-16 were found to be effective sperm additives, improving mitochondrial transmembrane potential (ΔΨm) and adenosine triphosphate (ATP) content, total antioxidant activity (T-AOC), superoxide dismutase (SOD) activity, and glutathione peroxidase (GPx) activity while reducing reactive oxygen species (ROS) levels, and malondialdehyde (MDA) content following preservation at 17 °C after a 5-day incubation. Both fluorescence and SEM showed that the serum exosomes bound directly to the sperm membrane, suggesting an interaction that could influence sperm-zona pellucida binding. Overall, this study provides new insights into the potential benefits of adding cbsExos and ssExos to enhance the quality of boar sperm during ambient temperature preservation, which may lead to advancements in sperm preservation strategies. [ABSTRACT FROM AUTHOR]

The quality of sperm significantly influences the reproductive efficiency of pig herds. High-quality sperm is necessary for efficient fertilization and to maximize the litter numbers in commercial pig farming. However, the understanding of genes regulating porcine sperm motility and viability is limited. In this study, we validated porcine sperm/Sertoli-specific promoters through the luciferase reporter system and identified vital genes for sperm quality via loss-of-function means. Further, the shRNAs driven by the ACE and SP-10 promoters were used to knockdown the SPAG6 and PPP1CC genes which were provisionally important for sperm quality. We assessed the effects of SPAG6 and PPP1CC knockdown on sperm motility by using the sperm quality analyzer and flow cytometry. The results showed that the ACE promoter is active in both porcine Sertoli cells and sperms, whereas the SP-10 promoter is operating exclusively in sperm cells. Targeted interference with SPAG6 and PPP1CC expression in sperm cells decreases the motility and increases apoptosis rates in porcine sperms. These findings not only offer new genetic tools for targeting male germ cells but also highlight the crucial roles of SPAG6 and PPP1CC in porcine sperm function. • Identified porcine germ cell-specific promoters ACE and SP-10. • Constructed shRNA vectors targeting SPAG6 and PPP1CC driven by ACE and SP-10 promoters. • Interfere with SPAG6 and PPP1CC resulting in decreased sperm motility and increased apoptosis. [ABSTRACT FROM AUTHOR]

Artificial insemination (AI) with liquid-preserved stallion semen is a widely used reproductive technology. As the demand for AI doses of high-class stallions is transnational, they are frequently exposed to long-distance transport. Since recent studies in boars indicated that vibration emissions caused by transport negatively affected sperm quality in vitro , this study questioned whether sperm quality in stallions is similarly impaired. Furthermore, we investigated stallion and extender-related differences in the spermatozoa's resistance to transport-related quality loss. Stallion ejaculates (n = 30) were collected at a German AI center, split in half, and subsequently diluted to a final sperm concentration of 50 × 106 sperm/mL using the semen extenders EquiPlus or Gent (both Minitüb GmbH, Germany). Four 12 mL aliquots of each sample were filled in plastic syringes according to a split-sample design and exposed to vibration (Displacement index D i = 3.0 ± 0.1) at 5 °C for 0 h (control), 3 h, 6 h or 9 h. All samples were stored for four days at 5 °C after transport simulation and analyzed for total sperm motility, thermo-resistance, membrane integrity, and mitochondrial activity determined by flow cytometry as well as the pH. After calculating generalized linear mixed models for each sperm quality trait, a negative impact of the duration of transport simulation could be shown on total sperm motility (P = 0.001), thermo-resistance (P = 0.030), and the pH (P = 0.001). Simulated transport for 6 h and 9 h diminished sperm quality (P ≤ 0.01), with 9 h reducing thermo-resistance by 5 ± 2.2% points (PP) for EquiPlus and sperm motility by 2.2 ± 1.7 PP for Gent compared to the control group. In contrast, samples exposed to vibration for 3 h showed no decline in sperm quality (P > 0.05). The individual stallion influenced every semen trait (P < 0.05) and transport-related losses in sperm thermo-resistance of up to 15.9 PP were demonstrated. Furthermore, EquiPlus was superior to Gent in all semen assessments (P < 0.001). We conclude that in vitro sperm quality is impaired by vibration. As the quality loss depends on the transport time, we recommend keeping shipping time as short as possible especially for spermatozoa of stallions that are susceptible to vibration-induced sperm quality loss. • Transport-related vibrations affect the quality of AI doses in stallions. • Long transport times reduce sperm motility and thermo-resistance in stallions. • The ability of sperm to resist transport stress varies greatly between stallions. • There is an individual stallion effect influencing all sperm quality traits. • EquiPlus is superior to Gent over all investigated stallion sperm parameters. [ABSTRACT FROM AUTHOR]

Polystyrene microplastics (PS-MPs) are important carriers of pollutants in water. 17α-Methyltestosterone (MT) is a synthetic environmental endocrine disrupting chemical (EDC) with androgenic effects. To study the effects of PS-MPs and MT on zebrafish reproductive systems, zebrafish were exposed to 0 or 50 ng L−1 MT, 0.5 mg∙L−1 PS-MPs, or 50 ng∙L−1 MT + 0.5 mg∙L−1 PS-MPs for 21 d. The results showed that the different exposure reagents caused varying degrees of damage to the reproductive systems in zebrafish, with the extent of damage increasing as the exposure duration increased. Histological analysis of the gonads revealed that the ratio of mature oocytes and mature spermatozoa in the gonad decreased gradually with increased exposure time, with the ratio being Control > PS-MPs > MT > MT + PS-MPs in decreasing order. The results of quantitative real-time PCR (qRT‒PCR) showed that in female fish treated for 7 d, the expression of cyp11a mRNA was significantly reduced in all three treatment groups(MT, PS-MPs, and MT + PS-MPs), while in the group treated for 14 d with MT + PS-MPs, the expression of cyp19a1a and StAR mRNA was significantly increased. In male fish exposed for 21 d, the expression of cyp11a , cyp17a1 , cyp19a1a , StAR , 3β-HSD , and 17β-HSD3 mRNA was significantly decreased in MT + PS-MPs. ELISA results showed that after 14 d of exposure, the levels of E2, LH, and FSH in the ovaries of female fish were significantly reduced in all three treatment groups. Similarly, the levels of T, E2, LH, and FSH in the testis of male fish were significantly reduced after 14 d of exposure to PS-MPs and MT + PS-MPs. Offspring of zebrafish exposed to MT and MT + PS-MPs exhibited delayed incubation time and slow development. The cross-generational toxicity of PS-MPs themselves may be negligible, but it can exacerbate the toxicity of MT, making the cross-generational effects more pronounced in the offspring, causing offspring mortality and malformations. Offspring of zebrafish exposed to MT and MT + PS-MPs exhibited delayed incubation time and slow development. In addition, MT caused malformations such as pericardial edema, yolk cysts, and spinal deformities in zebrafish during the incubation period. [Display omitted] • Both MT and MT + PS-MPs exhibited inhibitory effects on sperm and oocyte maturation. • -MT and MT + PS-MPs had a significant impact on the mRNA expression levels of steroid-related gene. • -MT and MT + PS-MPs led to a decrease in the levels of T, E2, LH, and FSH in the gonads of zebrafish. • -The progeny deformity rate in the MT and MT + PS-MPs groups was significantly higher than that in the control groups. [ABSTRACT FROM AUTHOR]

Taxifolin is a plant flavonoid effective as an antioxidant. This study aimed to assess the effect of adding taxifolin to the semen extender during the cooling period before freezing on the overall post-thawing sperm variables of Bermeya goats. In the first experiment, a dose-response experiment was performed with four experimental groups: Control, 10, 50, and 100 μg/ml of taxifolin using semen from 8 Bermeya males. In the second experiment, semen from 7 Bermeya bucks was collected and extended at 20 °C using a Tris–citric acid–glucose medium supplemented with different concentrations of taxifolin and glutathione (GSH): control, 5 μM taxifolin, 1 mM GSH, and both antioxidants. In both experiments, two straws per buck were thawed in a water bath (37 °C, 30 s), pooled, and incubated at 38 °C. Motility (CASA) was assessed at 0, 2, and 5 h, and sperm physiology was assessed at 0 and 5 h by flow cytometry (viability, intact acrosome membrane, mitochondria membrane potential, capacitation, intracellular reactive oxygen species —ROS—, mitochondrial superoxide, and chromatin status). In experiment 2, an artificial insemination trial (AI) was included with 29 goats for testing the taxifolin 5-μM treatment on fertility. Data were analyzed with the R statistical environment using linear mixed-effects models. In experiment 1 and compared to the control, T10 increased progressive motility (P < 0.001) but taxifolin decreased total and progressive motility at higher concentrations (P < 0.001), both post-thawing and after the incubation. Viability decreased post-thawing in the three concentrations (P < 0.001). Cytoplasmic ROS decreased at 0 and 5 h at T10 (P = 0.049), and all doses decreased mitochondrial superoxide post-thawing (P = 0.024). In experiment 2, 5 μM taxifolin or 1 mM GSH (alone or combined) increased total and progressive motility vs. the control (P < 0.01), and taxifolin increased kinematic parameters such as VCL, ALH, and DNC (P < 0.05). Viability was not affected by taxifolin in this experiment. Both antioxidants did not significantly affect other sperm physiology parameters. The incubation significantly affected all the parameters (P < 0.004), overall decreasing sperm quality. Fertility after artificial insemination with doses supplemented with 5 μM taxifolin was 76.9% (10/13), not significantly different from the control group (69.2%, 9/13). In conclusion, taxifolin showed a lack of toxicity in the low micromolar range and could benefit goat semen cryopreservation. • Supplementation of antioxidant compounds to semen extender: taxifolin, glutathione. • Sperm quality, functionality, and fertility of the native Bermeya goat breed. • Taxifolin showed encouraging results for cryopreserving goat semen. • Use of frozen semen and artificial insemination in goats. [ABSTRACT FROM AUTHOR]

Sperm preservation is an efficient technique used for the recovery, conservation, and management of some endangered fish species. The present study was conducted to explore how preservation time would affect sperm and spawning performance in the endangered delta smelt (Hypomesus transpacificus). Sperm were preserved with the modified Hanks balanced salt solution at 14.7–16.9 °C. The Kruskal-Wallis test of sperm parameters using OpenCASA plugin in ImageJ software showed that sperm (n = 33♂) had significantly higher velocity and motility within the first 5 s after activation than that of other time points, while sperm had the lowest velocity and motility after 3 min post activation (P < 0.001). The findings (n = 30♂) also showed fresh sperm had higher velocity and motility than preserved sperm, while the sperm preserved for over 24 h showed a significantly low performance (P < 0.001). The nonlinear mixed effects models of fertilization results (n = 14♂ × 70♀) indicated the fresh sperm and sperm preserved for 1 h had higher fertilization rates than other preservation times (P < 0.001). The hatching rate (n = 14♂ × 70♀) also showed the fresh sperm and sperm preserved for 3 min and 1 h had higher hatching rates than other preservation times (P < 0.001). Overall, the study showed the best sperm performance in delta smelt was found within the first 5 s post activation, and the best fertilization and hatching rates were found when the sperm were fresh and preserved for 1 h. The findings of this study provide information for the first time about how long the delta smelt's sperm are motile for quality analysis, and how the preservation time can affect sperm quality, fertility, and hatching of this species for future applications. • Delta smelt sperm has the highest motility within the first 5 s after activation. • Fresh sperm has higher motility than the sperm preserved in the extender tested. • Best fertilization and hatching rates were found in fresh sperm and sperm preserved for 1 h. [ABSTRACT FROM AUTHOR]

Lyophilisation is an alternative method for sperm preservation. The aim of this study was to evaluate the effects of freeze-thawing (F/T) and freeze-drying (F/D) on the quality of epididymal goat sperm. Sperm from each region of the epididymis (caput, corpus and cauda) were collected and evaluated for the expression of phospholipase C zeta (PLC-ζ), protamine 1 (PRM1), transition protein 1 (TNP1) and 2 (TNP2). The effects of F/T and F/D on sperm quality in terms of PLC-ζ expression, chromatin stability (Chromomycin A3; CMA3) and DNA integrity were examined. The fertilising ability after intracytoplasmic sperm injection (ICSI) was also tested. Fresh sperm existed PLC-ζ, PRM1, TNP1 and TNP2, irrespective of the regions of the epididymis. However, different patterns of PLC-ζ expression were found. Although PRM1, TNP1, TNP2 were still expressed after F/T or F/D, only F/T could preserve the presence of PLC-ζ. For fresh sperm, caput epididymal sperm had the lowest evidence of chromatin stability when compared to sperm harvested from other regions of the epididymis. The F/T and F/D further increased the numbers of CMA3-positive sperm (P < 0.001). In all cases, no CMA3 staining was observed in caudal epididymal sperm. The caudal epididymal sperm had significantly greater proportions of sperm with intact DNA compared with caput and corpus epididymal sperm, especially when F/T and F/D were performed. The fertilisation rates of F/D sperm tended to decrease when compared with F/T sperm (4.2 ± 3.2 vs. 13.6 ± 9.0, P = 0.08). It is concluded that the sperm recovered from the caudal epididymis is suitable for freezing and lyophilisation. However, poor fertilisation rates of F/D sperm were coincidently observed, with a deficit demonstration of PLC-ζ. • Cauda epididymal spermatozoa are suitable for freeze-drying compared with sperm from other regions of epididymis. • Freeze-drying disrupts PLC-ζ expression, sperm quality and fertilising ability of epididymal sperm. • Freeze-dry cauda epididymal spermatozoa could fertilise MII goat oocytes following ICSI and sperm activation. [ABSTRACT FROM AUTHOR]

This study aimed to investigate the effect of dexamethasone (DEX) on chemotaxis and phagocytic activity of neutrophils for spermatozoa and semen quality of preserved boar semen. The different concentrations of dexamethasone were added to boar semen dilutions to detect its effects on the chemotaxis of neutrophils and phagocytosis of neutrophils and sperm motility sperm malformation rate, plasma membrane integrity, mitochondrial activity, and spermatozoa motility parameters. The study results showed that the experimental groups of DEX significantly inhibited the phagocytosis and chemotaxis of PMNs for spermatozoa. With the increased concentration of DEX, there was an inhibition effect on PMNs activity. In addition, under 17 °C storage conditions, the addition of DEX 1 × 10−6 mol/mL concentration has the best preservation effect on boar semen, which can effectively improve the sperm motility, movement parameters, plasma membrane integrity, mitochondrial activity, T-AOC activity, and significantly reduce the sperm malformation rate and H 2 O 2 content. Therefore, the most suitable concentration of DEX to preserve boar semen at 17 °C is 1 × 10−6 mol/mL. The significant increase of conception rate of sows and litter size of piglets proved that DEX has practical application value. Thus, it is shown that the use of DEX to inhibit uterine inflammation is effective and feasible for sperm damage providing new methods for developing low-dose artificial insemination technology. • Dexamethasone (DEX) effectively reduced phagocytic activity and chemotaxis activity of PMNs induced by LPS for boar sperm. • DEX treatment (1 × 10−6 mol/mL) effectively delayed the decline of sperm motility, maintained sperm acrosome integrity, and improved sperm mitochondrial activity during storage. • DEX (1 × 10−6 mol/mL) played a positive role in improving the breeding performance of sows. [ABSTRACT FROM AUTHOR]

During cryopreservation, sperm encounters oxidative stress induced by excessive reactive oxygen species (ROS), destroying the sperm plasma membrane structure and reducing its physiological functions. The present study aimed to evaluate the effect of Astragalus polysaccharides (APS) on the cryopreservation of dairy goat semen. Semen was collected from six goats, and then qualified semen with movement >80% was selected after preliminary evaluation. The semen was divided into six aliquots, diluted with dairy goat semen extender (1:10) at 37 °C, containing 0 g/L (control), 0.1 g/L, 0.2 g/L, 0.3 g/L, 0.4 g/L and 0.5 g/L APS, cryopreserved, and stored in liquid nitrogen (−196 °C). Sperm quality was assessed after freeze-thawing. The highest sperm motility, motion performance, plasma membrane integrity, acrosome integrity, and antioxidant properties (total antioxidant capacity and levels of antioxidant enzymes) were recorded (P < 0.05) in the 0.2 g/L APS group after the semen was freeze-thawed. The control and the optimal group (0.2 g/L) were then selected to analyze the effects of APS on sperm energy metabolism (mitochondrial membrane potential [MMP] and adenosine triphosphate [ATP]), sperm apoptosis, and the expression of the AMPK signaling pathway. The results showed that treatment with 0.2 g/L APS increased sperm MMP and ATP content after freeze-thawing, reduced sperm apoptosis by regulating apoptosis-related proteins, and promoted AMPK phosphorylation by activating the AMPK signaling pathway. The cleavage rate of frozen goat sperm during in vitro fertilization (IVF) was also observed to increase. These findings suggest meaningful ways to improve cryopreservation of dairy goat semen and provide new insights into the mechanism by which APS protects sperm from oxidative damage via AMPK activation. • Astragalus polysaccharide (APS) improved motility, motion performance, acrosome integrity and plasma membrane integrity of dairy goat sperm after freezing and thawing, and the optimal concentration was 0.2 g/L. • APS increases the antioxidant capacity of sperm after freezing and thawing, and the optimal concentration was 0.2 g/L. • 0.2 g/L APS increased the sperm mitochondrial membrane potential and ATP content after freezing-thawing, reduced sperm apoptosis rate by regulating apoptosis-related proteins, and promoted sperm AMPK phosphorylation through activating AMPK signaling pathway. • 0.2 g/L APS increased the cleavage rate of goat frozen sperm in vitro fertilization (IVF) was increased. [ABSTRACT FROM AUTHOR]

Over the last decades, selection in cattle has mainly been based on milk production rather than on reproductive efficiency. While, when applied, focus on reproduction has involved females, attention has barely been paid to males and, if so, it has only looked at classical sperm quality parameters. In effect, variables such as telomere length have been missed, despite the fact that longer telomeres have been suggested to be linked to male fertility in humans. For this reason, the present study aimed to determine the length of telomeres in bovine sperm and their relationship with a) sperm quality evaluated through the conventional spermiogram and flow cytometry, and b) bull reproductive performance. For this purpose, 29 bulls were involved in this study. Sperm telomere length was evaluated through quantitative Fluorescent In Situ Hybridization (qFISH), and sperm quality was determined at 0 h and 4 h post-thaw. Bull fertility was assessed as non-return to estrus rates after 90 days of artificial insemination. Although the mean telomere length in bovine sperm was 12.06 ± 2.75 kb, the intra-individual variability in length led us to observe three different groups of telomeres in each sperm cell: short telomeres (7.14% ± 5.79% of telomeres; 8.29 ± 2.34 kb), medium telomeres (31.03% ± 12.92% of telomeres; 16.00 ± 2.72 kb) and long telomeres (61.93% ± 18.11% of telomeres; 30.13 ± 11.35 kb). Moreover, whereas reactive oxygen species (ROS) were found to be correlated to sperm telomere length (Rs = −0.492; P= 0.007), no correlation with other sperm quality parameters was found (P > 0.05). Reproductive performance after artificial insemination was not seen to be correlated to sperm telomere length (Rs = 0.123; P= 0.520). In conclusion, this study determined, for the first time, the mean telomere length in bovine sperm and also reported that there is a high variability within each sperm cell. Yet, while telomere length was found to be correlated to ROS generation, it was not related to bull reproductive performance. • Different telomere length subpopulations exist in bull sperm, as assessed by a clustering analysis of qFISH data. • Telomere length is related to the production of reactive oxygen species in bull. • Sperm telomere length is not an indicator for bull reproductive performance. [ABSTRACT FROM AUTHOR]

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 improved testicular hemodynamics by reducing the resistance of supratesticular artery. • Melatonin mitigated effects of heat stress (HS) on motility, sperm abnormalities and acrosome integrity from Weeks 2–7. • No differences between MEL and CONT in scrotal subcutaneous temperatures in the 4-d intervals before, during and after HS. • Melatonin has potential for mitigating effects of testicular HS under field conditions in rams. [ABSTRACT FROM AUTHOR]

This study aimed to assess 1) the effect of high environmental temperatures on sperm production and 2) the effectiveness of a temperature-humidity index (THI) to predict the degree of thermal stress in a cat model. Semen collection was performed by electroejaculation for 18 mo in 20 tomcats maintained under controlled photoperiod. Still, temperature and humidity were not experimentally manipulated to describe the effect of natural climate conditions on seminal samples. Ejaculates (n = 512) were then grouped according to temperature records of the sampling day and compared by temperature and THI index. Significant lower sperm parameters and increase sperm tail abnormalities were observed during warm environments (temperature and THI). Concentration and total sperm count were the most affected parameters. Environmental temperatures of 28.5 °C with 54% relative humidity (THI = 77.07) and 27.9 °C with 66% humidity (THI = 77.84) were upper thresholds of moderate thermal stress. Moreover, days with relative humidity near 90% led to severe thermal stress with temperatures as low as 26.6 °C (THI = 78.88). The current study demonstrates the detrimental effect of high environmental temperatures on sperm quality in the domestic cat. This effect is observed at lower temperatures when high relative humidity is present. In this sense, the THI was a reliable predictor of the magnitude of thermal stress experienced by cats. Thus, cats from reproductive programs should be maintained under controlled photoperiod cycles with temperatures around 20 °C and humidity around 70% to avoid semen detrimental effects. • Environmental temperatures and relative humidity affect sperm quality in toms. • A temperature-humidity index was used to study variations in sperm quality in toms. • When THI is over 72 a detrimental effect on seminal quality was observed. [ABSTRACT FROM AUTHOR]

Eurasian perch is an important fish species for European aquaculture diversification, but the quality of reproduction still remains one of the main limitations for further industry development. In particular, the optimal condition to obtain the best quality of sperm is poorly understood. The aim of our study was to measure the possible effects of two experimental rearing temperatures (6 °C and the conventionally used 12 °C) and of hormonal stimulation, on the motility parameters (pMOT, VCL, VSL, LIN, ALH, BCF), osmolality and fertilizing capacity of Eurasian perch sperm at the end of the reproductive cycle. A prior untested, large-scale (5 mL cryotube and Polystyrene box) cryopreservation method was implemented using fresh sperm obtained from the two above mentioned temperature groups. Males were injected with 100 μg body weight kg−1 sGnRHa. No significant difference was recorded between the two rearing temperatures and between the saline control and sGnRHa treated groups on the different features of sperm quality. A similar fertilization rate was monitored in all sGnRHa treated (6 °C: 69 ± 13%, 12 °C: 81 ± 11%) and saline control groups (6 °C: 79 ± 10%, 12 °C: 87 ± 4%). Correspondingly, no significant difference in hatching rate was observed in the sGnRHa injected (6 °C: 27 ± 9%, 12 °C: 40 ± 20%) and saline control (6 °C: 35 ± 18%, 12 °C: 36 ± 7%) males. However, a notable negative effect of freezing process on sperm movement was observed following thawing in both temperature groups. No significant difference in the motility parameters was measured between the two temperature groups following large-scale cryopreservation. Furthermore, a similar result was observed in the fertilizing capacity (6 °C: 79 ± 10%, 12 °C: 75 ± 8) of thawed sperm as well as in the hatching rate (6 °C: 52 ± 13%, 12 °C: 46 ± 19%). Our results indicate that fresh Eurasian perch sperm can tolerate a reduced rearing temperature following hormonal treatment. The adopted large-scale cryopreservation method could be used efficiently in the future for the fertilization of large amounts of Eurasian perch eggs following a precise standardization process. • No significant difference was recorded between the two rearing temperatures on the different features of sperm quality. • A similar fertilization and hatching rate was monitored in all sGnRHa treated and saline control groups. • No significant difference in the motility was measured between the two temperature groups following cryopreservation. • A similar high fertilization and hatching rate were measured by the two rearing temperatures using cryopreserved sperm. [ABSTRACT FROM AUTHOR]

Incorporation of Coenzyme Q10 (CoQ10) to the freezing medium provides advantageous effect for sperm cryopreservation in a variety of animal species, yet which has not been tested in giant grouper (Epinephelus lanceolatus). This research was designed to elucidate if CoQ10 could be used as a potential additive to improve giant grouper sperm quality after cryopreservation. After the process of freezing and thawing, various sperm quality parameters including motility, viability, apoptosis, mitochondrial membrane potential (MMP), intracellular reactive oxygen species (ROS) generation, DNA fragmentation as well as fertilization rate were evaluated with CoQ10 added at concentrations of 0, 25, 50 and 100 μM. Compared to the control group (0 μm), addition of CoQ10 in the medium yielded significantly higher total motility and curvilinear velocity, whereas the progressive motility, straight-line velocity and average path velocity were not differ from each other. An obvious improvement in viability was observed in spermatozoa cryopreserved with 25 and 50 μM CoQ10, while the apoptosis rate in CoQ10 treated groups (25, 50 and 100 μM) exhibited significantly lower values than that of the control. Besides, the production of ROS was significantly decreased with CoQ10 addition groups when compared with the control. In consistent with the improvement in antioxidant defense, CoQ10 supplementation in the medium also enhanced mitochondrial activity and reduced DNA fragmentation. In addition, freezing medium supplemented with CoQ10 also improved the fertilization success, a significantly higher fertilization rate was recorded at the concentration of 50 μM, but this value was not differ from that of 25 μM. Overall, the antioxidant CoQ10 provided an obvious beneficial effect on post-thaw quality of giant grouper spermatozoa. It was concluded that the optimal concentration of CoQ10 is 50 μM in the freezing medium. • Freezing medium supplemented with Coenzyme Q10 (CoQ10) attenuates oxidative stress for frozen-thawed giant grouper sperm. • CoQ10 had an obvious beneficial effect on post-thaw quality and fertility of giant grouper sperm. • The optimal concentration of CoQ10 is 50 μM in the freezing medium. [ABSTRACT FROM AUTHOR]

The study aimed to evaluate if the sperm telomere length can be considered as a new biomarker for sperm quality in bulls. Sperm Telomere Length was evaluated by Monochrome Multiplex Quantitative PCR in group A (n = 8) and group B (n = 8) bulls, classified according to standard semen analysis. Also, this parameter was measured before and after Percoll gradient separation within bulls that produced semen of satisfactory quality. Sperm telomere length, measured as T/S ratio (average ratio of telomere repeats copy number to a single copy gene), was higher in group A than in group B bulls (0.77 ± 0.03 vs 0.43 ± 0.06; P < 0.01). Sperm telomere length was positively correlated with motility, viability and membrane integrity, and it was negatively correlated with sperm anomalies. Furthermore, Percoll gradient selected sperms with higher T/S ratio than unselected sperms (1.19 ± 0.02 vs 0.67 ± 0.03). These results suggest that sperm telomere length can be used as a new marker of bovine semen quality. • Sperm telomere length can be used as marker of bovine semen quality. • Sperm telomere length is correlated to most sperm quality traits. • Percoll density gradient separates sperm with longer telomeres. [ABSTRACT FROM AUTHOR]

The microbiome plays a key role in homeostasis and health and it has been also linked to fertility and semen quality in several animal species including swine. Despite the more than likely importance of sperm bacteria on the boar's reproductive ability and the dissemination of pathogens and antimicrobial resistance genes, the high throughput characterization of the swine sperm microbiome remains scarce. We carried RNA-seq on 40 ejaculates each from a different Pietrain boar and found that a proportion of the sequencing reads did not map to the Sus scrofa genome. The current study aimed at using these reads not belonging to pig to carry a pilot study to profile the boar sperm bacterial population and its relation with 7 semen quality traits. We found that the boar sperm contains a broad population of bacteria. The most abundant phyla were Proteobacteria (39.1%), Firmicutes (27.5%), Actinobacteria (14.9%) and Bacteroidetes (5.7%). The predominant species contaminated sperm after ejaculation from soil, faeces and water sources (Bacillus megaterium , Brachybacterium faecium , Bacillus coagulans). Some potential pathogens were also found but at relatively low levels (Escherichia coli , Clostridioides difficile , Clostridium perfringens , Clostridium botulinum and Mycobacterium tuberculosis). We also identified 3 potential antibiotic resistant genes from E. coli against chloramphenicol, Neisseria meningitidis against spectinomycin and Staphylococcus aureus against linezolid. None of these genes were highly abundant. Finally, we classified the ejaculates into categories according to their bacterial features and semen quality parameters and identified two categories that significantly differed for 5 semen quality traits and 13 bacterial features including the genera Acinetobacter , Stenotrophomonas and Rhodobacter. Our results show that boar semen contains a bacterial community, including potential pathogens and putative antibiotic resistance genes, and that these bacteria may affect its reproductive performance. • A pilot RNA-seq study in 40 Pietrain ejaculates to characterize the porcine sperm microbiome. • Most bacteria in our pig sperm samples were contaminants from soil, water and faeces. • Potential pathogens were scarce and the most abundant was Escherichia coli. • Resistance genes against chloramphenicol, spectinomycin and linezolid were predicted. • LINK-HD draw two sample clusters defined by 13 bacterial features and 5 semen traits. [ABSTRACT FROM AUTHOR]

Since bull fertility may be adversely affected by hot humid conditions, the current increase in global temperature is of concern for future livestock production. Heat stress occurs when the body's normal physiological mechanisms to regulate body temperature cannot cope with external conditions. The testes and scrotum have their own complex regulatory mechanisms to protect developing sperm during their most vulnerable stages, but even these may be overwhelmed by unfavourable external conditions. The effects of mild, moderate and severe heat stress are somewhat different, with cattle exposed to mild and moderate heat stress apparently showing an adverse effect on fertility, whereas cattle in very hot, humid climates almost continuously may not exhibit any difference in sperm quality throughout the year. This apparent paradox may be due to differences in the cattle populations being studied, since they could differ in breed, age, purpose (beef versus dairy), or even in the methods used to assess sperm quality. The adverse effects on fertility may occur through the effects of reactive oxygen species on sperm DNA, or through perturbation of the production of antioxidants that usually protect sperm from oxidative attack. These effects can be mitigated to some extent by choosing breed and age of bulls with care, and adopting breeding strategies that avoid semen collection or ejaculation at the most adverse times of year. Husbandry measures such as controlled ventilation, misting, provision of shade or cool surfaces for lying down, could aid temperature regulation. Avoiding heat stress during late pregnancy aids calf growth in early life; careful feeding regimens for young bull calves create good conditions for sperm quality after puberty. Bull fertility is too important to be left to chance. Breeds should be chosen according to climate conditions and the purpose of livestock production. • Bull fertility may be adversely affected by hot humid conditions. • Manifestation of heat stress represents a complex interaction of factors. • Effects of natural mild, moderate and severe heat stress differ. • Scrotal insulation is not a representative model for climate change. • Heat stress can be mitigated to some extent by management and husbandry. [ABSTRACT FROM AUTHOR]

Spermatozoa are diverse in form and function and these differences impact on their fertilizing capacity. Because of considerable inter-male and inter-species differences in sperm traits, assessments of sperm quality demand that we consider variations at different levels. We should thus pay attention not only to average values but also intra- and inter-sperm population variations and subpopulation structure. Sperm shape and size evolve in reponse to postcopulatory sexual selection. Assessments of morphological variation, with conventional microscopy or with computer-assisted systems, should bear this in mind. In rodents sperm head shape is asymmetric so it requires more complex tools, such as geometric morphometrics. Sperm function also evolves under postcopulatory sexual selection and this could be used as a basis to assess sperm performance. Sperm cells swim actively to overcome barriers in the female tract and develop a peculiar motility pattern in the final stages prior to and during fertilization. Both types of movement can be analyzed by computer-assisted microscopy systems. Sperm have high energetic demands for cell homeostasis, motility, and signalling. Bioenergetics can be analyzed by various means, including extracellular flux analyses to characterize glycolysis and mitochondrial respiration. Finally, cell signalling during capacitation has received much attention and can be assessed by microscopy (conventional or computer-assisted) or flow cytometry. Recent advances in image-flow cytometry affords analyses of high cell numbers with spatial localization of subcellular changes, which will have a big impact in the development of functional tests for the andrology clinic and in sperm preservation and use in artificial insemination. • This review focuses on several sperm traits that can be assessed to estimate fertilizing capacity. • Sperm morphology, swimming patterns, bioenergetics and cell signalling are considered. • Methods to examine sperm traits are reviewed in relation to fertility assessments. • Intra- and interspecific differences in sperm morphology and function are highlighted. [ABSTRACT FROM AUTHOR]

Laserpuncture is one of the applicative technologies used mainly in animal and fish reproductions. Laserpuncture technology has been used to improve gonadal maturity and sperm quality in fish rapidly. This study aimed to determine the effects of different laserpuncture doses on gonadal maturity and sperm quality of male striped catfish. Males striped catfish (800–900 g/fish body weight) and I gonadal maturity stage were used. Semi-conductor soft laser was used with doses of 0.2-, 0.4-, and 0.5-J, while the negative control (without the laserpuncture and the ovaprim™) and only the ovaprim™ were used as a comparison treatment, respectively. The soft-laser was treated on reproductive acupoint every week for four weeks, while the ovaprim™ was administered by intramuscular injection at dose of 0.2 mL/kg fish in final rearing period. Fish was reared in hapa at the controlled pond. Fish was fed with a commercial feed containing 32% crude protein. Gonadal maturity, gonadosomatic index (GSI), hepatosomatic index (HSI), and sperm quality of male striped catfish were measured in the final rearing period. The results showed that the laserpuncture on the reproductive acupoint had a highly significant effect (P < 0.01) on the gonadal maturity, GSI, HSI, and sperm quality of male striped catfish. In terms of the gonadal maturity, laserpuncture doses treatment of 0.4 and 0.5-J gave the most mature IV stage. While the highest levels of GSI and HSI were found in 0.5-J of laserpuncture dose, which was 2.17% and 1.54%, respectively. In addition, the best sperm qualities were observed in 0.5-J of laserpuncture dose, which were 81.75% motility, 82.75% viability, and 7.0 × 109 cell/mL concentration. These results suggest that the laserpuncture can accelerate a gonadal maturity and improve sperm quality in male striped catfish. • The low-power soft laser can be used to laserpuncture treatment. • Rapidly gonad mature can induce by laserpuncture treatment. • Laserpuncture stimulates spermatogenesis. • Laserpuncture improves sperm quality. • Gonadal maturity can be stimulated during the non-spawning season. [ABSTRACT FROM AUTHOR]

Sperm activation involves ion fluxes as well as a previous maturation in the seminal plasma, something which has not been studied in depth in marine fish species. pH and potassium are probably involved in sperm maturation and motility in the European eel, as indicated in previous studies. In this work, the absolute intracellular concentration of potassium in European eel sperm has been determined for the first time. In addition, the intracellular pH (pH i) of quiescent eel spermatozoa was determined by two methods (nigericin and null point) that gave similar results, 7.4–7.6. The natural pH i range of sperm samples in the quiescent stage was 7.4–8.0, with no evident relationship with sperm motility. However, a linear correlation was seen between sperm motility and the pH of the diluent or extracellular pH (pH e), as well as between the pH i and the pH of the diluent. The pH i change post-activation in seawater (ASW) depended on the initial pH e of the diluent medium. Activation with ASW induced an internal alkalinization of the cells when the sample had previously been diluted in a pH e < 8.0; an acidification when pH e > 8, and no pH i variation when pH e was 8.0. These experiments indicated that a careful selection of the diluents should be performed before measuring natural pH i changes in sperm cells. Thus, studies on the specific seminal plasma composition of marine fish species are necessary before studying their physiology. Furthermore, our study indicates that intracellular alkalinization is not a universal fact during sperm activation. • A gradient of potassium between the eel sperm cell and the seminal plasma has been demonstrated for the first time. • The intracellular pH of eel sperm cells oscillates between 7.4 and 8.0 in physiological conditions. • The intracellular pH of spermatozoa is linearly dependent on the extracellular pH. • The change in the intracellular pH of sperm cells at activation is dependent on the pH of the diluent medium. [ABSTRACT FROM AUTHOR]

The present work aimed at setting a new test of boar sperm functional competence (SFCt) to determine the association with sperm in vitro fertilizing ability and cryotolerance. Three independent experiments were conducted. In the first experiment, a gage repeatability and reproducibility (R&R) study was carried out to determine the reliability of the SFCt. Following this, 1565 ejaculates were clustered on the basis of sperm membrane and acrosome integrity, total motility, morphology and membrane functional integrity. Two groups were obtained and their SFCt values were compared. In the second experiment, 45 ejaculates were classified into two groups based on cleavage rates after in vitro fertilization and the SFCt outcomes of the two groups were examined. In the third experiment, 45 ejaculates were cryopreserved and classified as with good (GFE) or poor freezability (PFE) based on their post-thaw sperm membrane integrity and total motility; the SFCt outcomes in liquid-stored semen were also compared. ROC curves were used to address the accuracy of the SFCt in each experiment. In the R&R study, the greater variability of the study was attributed to the differences between ejaculates (97.61%); SFCt values were significantly (P < 0.01) higher in the good than in the poor sperm quality group. Ejaculates with high cleavage rates showed significantly (P < 0.05) higher SFCt values than ejaculates with low cleavage rates. SFCt values of GFE before cryopreservation were significantly (P < 0.05) higher than those of PFE. The SFCt had a fair discriminatory value in all experiments. In conclusion, the SFCt is a useful and reliable test to evaluate the sperm quality and is also related to the sperm resilience to withstand freeze–thawing procedures. However, further studies evaluating blastocyst formation and AI trials with a large number of boars are needed to confirm the accuracy of this test. • A new test based on the hypotonic resistance of boar spermatozoa has been set (SFCt). • The R&R study and screening of 1565 ejaculates confirmed the reliability of the SFCt. • The SFCt may provide more information than the separate conventional parameters. • The SFCt is associated with the in vitro fertilizing ability of a given ejaculate. • The SFCt is associated to the sperm resilience to withstand freeze-thawing. [ABSTRACT FROM AUTHOR]

Abstract Oxidative stress has been known as a significant cause of the lower fertility rates correlated with liquid stored rooster semen. The effect of coenzyme Q10 (CoQ10), as a powerful antioxidant, seems be beneficial on semen storage of broiler breeder roosters at the cooled condition. Therefore, two experiments were performed to assess the effect of CoQ10 supplemented semen extender on sperm quality parameters, fertility, hatchability and sperm penetration (SP) rates of rooster semen stored at 5 °C. In the first experiment, semen samples of 12 roosters were weekly pooled for four weeks (47–50 weeks of age). The pooled semen was diluted by modified Beltsville poultry semen extender and divided into three equivalent parts containing different levels of CoQ10 [0 (Q-0), 100 (Q-100) and 200 (Q-200) μM/mL) and then stored for 24 h at 5 °C. Sperm quality including progressive motility, plasma membrane integrity and functionality were evaluated after 0 and 24 h storage. The results showed that progressive motility, plasma membrane integrity and functionality were improved in Q-200 compared to Q-0 after 24 h storage at 5 °C (P < 0.01, P < 0.05 and P < 0.01, respectively). According to the results of the first experiment, Q-200 group was selected to be used to evaluate the fertility, hatchability and SP rate in the second experiment during next four weeks (51–54 weeks of age). The results of the second experiment showed that fertility rate was significantly increased in Q-200 compared to control group by approximately 10%, although no significant difference was observed in hatchability and SP rates between Q-200 and control groups. In conclusion, the results of the present research confirm that supplementation of rooster semen extender with CoQ10 might be potentially used to improve semen quality and fertility rates. Highlights • We tested the effect of adding CoQ10 to rooster semen extender on sperm quality stored at 5 °C for 24 h. • Addition of CoQ10 to extender improves sperm quality. • 200 μM/mL of CoQ10 appears to be optimal dose for fertility rate of rooster sperm following artificial insemination. [ABSTRACT FROM AUTHOR]

Abstract This study focused to determine beneficial impact of feeding quercetin supplemented diet on semen quality in summer heat imposed rabbits. Twelve heat stressed (HS) adult rabbits bucks were either fed with basal diet (HS; n = 06) or quercetin supplemented diet (QU-HS; n = 06) for a period of 56 days. Semen samples were collected and evaluated for volume, osmolality, morphology, concentration, motility, motion kinetics, viability, acrosome integrity, mitochondrial potential, and seminal plasma MDA level. Semen volume, concentration, motility and sperm kinetics parameters were affected by diet supplementation. Diet affected the sperm mitochondrial potential and day of treatment affected the viable sperm percentage. There was an effect of diet, day of treatment and diet by day interaction on acrosome reaction rate. Sperm head abnormalities were influenced by diet provision, sperm mid-piece abnormalities were affected by diet and day of treatment, whereas, the effect of diet and diet by day of treatment interaction were observed for total sperm abnormalities. There was an effect of diet and diet by day interaction for seminal plasma MDA level. In conclusions, quercetin reduces the damaging effects of HS and maintains the semen quality by lowering the oxidative stress in rabbits. Highlights • Heat stress affects the sperm quality in exposed male rabbits. • Dietary intake of quercetin maintains the semen quality in rabbit. • Quercetin provision the lower the HS induced damages by reducing the oxidative stress. [ABSTRACT FROM AUTHOR]

Abstract Reproductive defects can occur when the integrity of the male gamete genome is affected. Sperm chromatin is not homogeneous, having relaxed regions which are more accessible to the transcription machinery in the embryo, and thought to be specially sensitive to DNA damage. The level of damage in specific genes located in these sensitive regions could represent an early biomarker of damage. Our objective is to test the hypothesis that these more relaxed regions show greater susceptibility to damage in zebrafish, a species lacking protamines and whose sperm chromatin is compacted with histones. After sperm UV irradiation, treatment with H 2 O 2 and cryopreservation, global chromatin fragmentation was evaluated using the TUNEL assay, and the number of lesions per 10 Kb in specific genes (hoxa3a , hoxb5b , sox2, accessible for early transcription and rDNA 18S and rDNA 28S) was quantified by using a qPCR approach. Additionally, oxidative damage within the sperm nucleus and the potential colocalization of this injury with histone H3 and TOPO IIα+β were located by using immunofluorescence. UV irradiation produced the highest degree of fragmentation (p = 0.041) and the highest number of lesions per 10 Kb in all the genes, but no differences were observed in sensitivity to damage in the studied genes (ranging from 14.93 to 8.03 lesions per 10 Kb in hoxb5b and 28S, respectively). In contrast, H 2 O 2 and cryopreservation caused varying levels of damage in the analyzed genes which was not related to their accessibility, ranging from 0.00 to 1.65 lesions per 10 Kb in 28S and hoxb5b , respectively, after H 2 O 2 treatment, and from 0.073 to 5.51 in 28S and sox2 , respectively, after cryopreservation. Immunodetection near oxidative lesions also revealed different spatial patterns depending on the treatments used, these being mostly homogeneous with UV irradiation or cryopreservation, and peripherally located around the nucleus after H 2 O 2 treatment. Oxidative lesions did not colocalize with histone H3 or TOPO IIα+β, thus demonstrating that the relaxed DNA regions associated with these proteins were not more vulnerable to oxidative damage. Results suggest that accessibility of each agent to the nucleus could be the main factor responsible for the distribution of sperm DNA damage rather than the organization of the chromatin. Lesions in these genes important to early embryo development assayed in this study cannot be used as biomarkers of global DNA damage. Highlights • Susceptibility to damage varies among genes in zebrafish sperm. • Neither chromatin packaging nor epigenetic pattern determines differential gene susceptibility. • Distribution of oxidative DNA damage varies among treatments and does not colocalize with histone H3 or with TOPO IIα+β. • None of the analyzed genes are sensitive to a range of genotoxic treatments and cannot be used as sperm quality biomarkers. [ABSTRACT FROM AUTHOR]

Abstract Two experiments were conducted to evaluate the effects of antibiotic-containing extender of on sperm quality and control of bacterial growth. In Experiment 1, ejaculates were diluted in extender containing no antibiotics, potassium penicillin G-amikacin disulfate (PEN-AMIK), ticarcillin disodium-potassium clavulanate (TICAR-CLAV), piperacillin sodium/tazobactam sodium (PIP-TAZ), or meropenem (MERO). In freshly extended semen, only slight differences were detected among some antibiotic treatments for total sperm motility, curvilinear velocity, and viable acrosome-intact sperm (P < 0.05). In cool-stored semen, slight differences were also detected among certain antibiotic treatments for curvilinear velocity and chromatin integrity (P < 0.05). In Experiment 2, ejaculates were diluted in extender and subjected to no bacterial spiking, or inoculated with lower or higher doses of K. pneumoniae or P. aeruginosa. Following cooled storage of semen, colony forming units/ml (CFU/mL) were less in PEN-AMIK (706 ± 244) and MERO (1576 ± 1076) treatment groups than in TICAR-CLAV (4678 ± 1388) or PIP-TAZ (8108 ± 3198) treatment groups (P < 0.05). The CFU/mL were lower in all antibiotic-containing treatment groups than the control group (18478 ± 4374; P < 0.05). The percentage of culture plates containing no bacterial growth in unspiked semen was greater in PEN-AMIK (75%) than PIP-TAZ (15%) or TICAR-CLAV (20%; P < 0.05). The percentages of culture plates containing no bacterial growth in semen spiked with a lower doses of K. pneumoniae or P. aeruginosa were higher in PEN-AMIK (70% and 50%, respectively) then in all other treatment groups (0–40% and 0–15% for K. pneumonia and P. aeruginosa , respectively; P < 0.05); however, complete control of bacterial load was only modest even with PEN-AMIK. In both experiments, freezing and thawing extender prior to use did not have any appreciable detrimental effect on sperm quality or antibiotic efficacy. In summary, all antibiotics tested had minimal effects on measures of sperm quality in fresh or cool-stored semen extenders; however, PEN-AMIK, followed by MERO, yielded the best results in terms of antimicrobial efficacy. None of the antibiotic types controlled bacterial growth, in comparison with the antibiotic-free control group, when extended semen was spiked with a high concentration of Pseudomonas aeruginosa. Cooled storage of extended semen reduced bacterial growth in comparison with freshly extended semen. Highlights • None of the antibiotic-containing extenders tested had a demonstrable negative effect on stallion sperm quality. • Lowest bacterial counts were detected in extended semen containing penicillin/amikacin or meropenem. • Highest total antibacterial effectivity was obtained in extender containing penicillin/amikacin. [ABSTRACT FROM AUTHOR]

The purpose of this study was to investigate the effects of ram melatonin treatment on the sperm quality and metabolite composition of the seminal plasma in the non-breeding season. Four mature rams were treated with 54 mg melatonin in March subcutaneous implants and four untreated rams served as control. At 0, 30, 90 and 120 days semen samples were collected and sperm, separated from seminal plasma, was evaluated for its capacity to fertilize and produce embryos in vitro. Seminal plasma metabolites were extracted and analyzed by capillary electrophoresis/mass spectroscopy. In the resulting electropherograms, the area corresponding to selected metabolites was extracted and quantified. Ram melatonin treatment affected the in vitro fertilization competence of sperm. Blastocyst output increased until 90 days after treatment (27.20 ± 7.35 vs 54.7 ± 4.4% at 0 and 90 days respectively; p < 0.05) while the untreated group did not show statistical differences. In treated rams, the concentration of melatonin in seminal plasma increased from 3.34 ± 1.70 at day 0–9.65 ± 2.89 AU (Arbitrary Units) after 90 days, then decreased to reach the level of the untreated ram after 120 days (p < 0.05). During 90 days after melatonin treatment, an increase (p < 0.05) in seminal plasma concentrations of glutamic acid (6.28 ± 1.53 vs 14.93 ± 1.53 AU at 0 and 90 days respectively), glutamine (16.89 ± 4.65 vs 54.51 ± 4.65 AU), carnitine (22.97 ± 9.81 vs 104.30 ± 9.81 AU), acetyl-carnitine (48.15 ± 17.32 vs 217.69 ± 17.32 AU), choline (1.82 ± 1.55 vs 14.16 ± 1.55 AU) and arginine (1.31 ± 1.08 vs 14.25 ± 1.08 AU) was detected. Tyrosine concentration increased during 30 days from melatonin treatment (12.79 ± 3.93 vs 27.08 ± 3.04 AU) but at 90 days its levels were similar to the untreated group. In conclusion, melatonin treatment during the non-breeding season improves the concentration of several metabolites in seminal plasma and sperm fertilization competence in Sarda breed ram. [ABSTRACT FROM AUTHOR]

SexedULTRA ™ is an improved method of sex-sorting sperm creating a less damaging environment to retain sperm integrity through the sorting process. The aim of this study was to evaluate the in vitro characteristics of fresh and frozen bovine sperm using the SexedULTRA ™ method, and compare it to conventional (non-sorted) sperm. For both methods, percent total sperm motility was estimated visually and also classified into total and progressively motile using a computer assisted sperm analyzer (CASA). Percent sperm with intact plasma membranes (VIA) and acrosomes (PIA) were assessed using flow cytometry and sperm DNA fragmentation index (DFI) was estimated using the Bull sperm Halomax ® Kit. Two contemporaneous ejaculates from 10 bulls were processed and cryopreserved using one of the two procedures (SexedULTRA ™ and conventional). Sperm motility, VIA and PIA were assessed post-thaw (0 h) and post-incubation (3 h at 37 °C, 8 h and 24 h at 18 °C). DFI was analyzed post-thaw (0 h) and after 6, 24, 48 and 72 h of incubation at 37 °C. In a second experiment, ejaculates from 7 bulls were split sampled into the two types of processing (SexedULTRA ™ and conventional) and diluted using a fresh semen extender developed for sex-sorted bovine sperm. Sperm quality was assessed after dilution (0 h) and after incubation for 12, 24, 48, 72 h at 18°, and the same time points of incubation at 37 °C for DFI. Frozen-thawed SexedULTRA ™ sperm was significantly (P < 0.05) better than conventional semen after a 3 h incubation at 37 °C for PIA, and after a 24 h incubation at 18 °C for percent visual motility and PIA. DFI was significantly lower for SexedULTRA ™ compared to conventional at all time points of incubation (37 °C). Fresh SexedULTRA ™ sperm showed improved quality compared to conventional at all time points of incubation at 18 °C for percent visual and total motile sperm, VIA, PIA, and DFI. Significant differences were also found in progressive motile sperm immediately after dilution (0 h), but not at any time point after incubation. The results show that the SexedULTRA ™ process maintains the quality of sex-sorted sperm and, in many cases, has better in vitro longevity than conventional semen. [ABSTRACT FROM AUTHOR]

The present study evaluated whether two different antimicrobial peptides (AMP): porcine beta defensins-1 (PBD1) and −2 (PBD2) at three concentrations (1.5 μM, 3 μM and 5 μM) could be a suitable alternative to antibiotics in liquid-stored boar semen. Two separate experiments were conducted with liquid-stored boar semen preserved at 17 °C for 9–10 days. In the first one, we evaluated the impact of adding three concentrations of each AMP on the bacterial growth and sperm quality of boar semen stored for 10 days. In the second experiment, the ability of these AMPs to control bacterial growth was determined over a 9-day period, following artificial inoculation with Escherichia coli at 10 7 and 10 8  CFU mL −1 . In both experiments, sperm viability was assessed through flow cytometry, sperm motility was determined with Computer Assisted Sperm Analysis (CASA) and the inhibitory effect on microbial growth was evaluated by bacteria culture on Luria Bertani agar. PBD1 and PBD2 were found to significantly ( P  < 0.05) decrease sperm motility at 5 μM (% total motile spermatozoa at day 10, Control: 31.6% ± 1.2% vs. PBD1: 6.5% ± 0.3% and PBD2: 5.6% ± 0.4%). Although the highest inhibitory effect on bacterial growth was observed at 3 μM (day 10, PBD1: 1.4 × 10 6  ± 6.2 × 10 5  CFU mL −1 and PBD2: 9.1 × 10 5  ± 2.4 × 10 5  CFU mL −1 ) and 5 μM (day 10, PBD1: 1.2 × 10 5  ± 5.1 × 10 4  CFU mL −1 ; PBD2: 8.7 × 10 4  ± 2.9 × 10 4  CFU mL −1 ), the control with antibiotic was found to be more effective (day 10, 8.3 × 10 3  ± 1.6 × 10 3  CFU mL −1 ). In conclusion, PBD1 and PBD2 may be added to antibiotic-free extenders for boar semen at a concentration of 3 μM, but do not completely control all bacterial growth. [ABSTRACT FROM AUTHOR]

This study aimed to report in detail, the technique and challenges of cloacal massage, to collect and evaluate semen from red-winged tinamou ( Rhynchotus rufescens ) keep in captivity, performed by only one technician. Sixty-four semen collection attempts, from 16 adult males, during breeding season and 16 attempts form these same 16 males in non-breeding season, were performed. Prior to collection, all animals were conditioned to cloacal massage for 6 weeks and the ejaculates were succeed with viable spermatozoa and then, evaluated for feces, urine and mucus contamination, volume, concentration, sperm vigor, motility, morphological defects and acrosome integrity. Semen collection success rate was 63% in breeding season and 2 (5%) samples were discarded by grade 5 contamination. Only 3 ejaculates from 16 tinamou were obtained in non-breeding season. Sperm concentration and acrosome integrity was higher (p = 0.00) in breeding season, and the percentage of total sperm morphological defects, were high in both in breeding and out breeding season. Overall, we concluded that the red-winged tinamou breeding season, is linked to photoperiod (spring and summer), and at this period time, semen can be obtained by cloacal massage collection satisfactorily, allowing its use in reproduction biotechnologies and sperm cryopreservation. [ABSTRACT FROM AUTHOR]

Standardized evaluation of sperm quality is essential for research, commercial-scale cryopreservation, and induced spawning. However, standardized methods for evaluation of sperm bundles (spermatozeugmata or spermatophores) have not been established. The purpose of the present study was to use Redtail Splitfin ( Xenotoca eiseni ) as a model for freshwater livebearing fishes to establish initial standardized methods to collect sperm bundles, and quantitatively and qualitatively evaluate quality-related attributes. No sperm or sperm bundles were able to be collected by stripping. Testes were removed, rinsed, weighed, placed in 50 μL of buffer solution on a glass slide, and crushed gently 3–5 times with angled spade-tip forceps. Sperm bundles were released into the buffer solution and collected with a pipette into 1.5-mL centrifuge tubes. To quantify size and shape, images of bundles were captured with a CCD camera connected to a microscope, and measured with computer software. There was no significant correlation between body wet weight and major bundle axis length ( P = 0.6759), minor axis length ( P = 0.5658), average axis length ( P = 0.5869), aspect ratio ( P = 0.7839), and observed area ( P = 0.5727). The concentrations of sperm bundles, estimated with the three methods (Makler ® counting chamber, a hemocytometer, and direct counting) were significantly different ( P < 0.0001). Hemocytometers were suitable for estimation of bundles from X. eiseni . To evaluate activation of sperm, bundles were viewed with a microscope, and classified into one of five phases by evaluating morphology of the bundles and motion of sperm within the bundles as Phase 0 through Phase 4 that represented early through late activation stages. The frequencies and duration of each activation phase were used to evaluate dissociation of sperm bundles and motility capability of sperm within the bundles. Within 180 min of activation, all five phases were observed. Overall, this study for the first time established standardized methods to collect and evaluate quality-related attributes of sperm bundles. These standardized evaluations provide a basis for further modification, standardization, and generalization, which are useful in research on livebearing fishes involving male gametes, such as studies on cryopreservation, artificial insemination, and in development of germplasm repositories for imperiled species including goodeids. [ABSTRACT FROM AUTHOR]

In this study, semen samples were collected from 96 Sanjabi rams in order to investigate the IGF-1 gene polymorphisms and their relationship with the characteristics of semen quality and testicular size. The dimensions of scrotal length, width and circumference were measured during autumn and spring over two years. Blood samples were simultaneously collected from jugular vein to extract DNA. PCR was performed using specific primers to amplify 294 and 272bp fragments including 5′ regulatory region and exon 3 of IGF-1 gene, respectively. PCR products were digested by B FO I and E co 88l restriction enzymes, respectively. Two genotypes including AA (194 and 100bp), AB (294, 194 and 100bp) and all possible genotypes including CC (182 and 90bp), CT (272, 182, and 90bp) and TT (272bp) were observed for 5′ flanking region and exon 3 of IGF-1 gene, respectively. The significant differences among IGF-1 genotypes for testicular dimensions were not observed. However, the polymorphism of 5′ flanking region in the studied population had significant effect on individual motility and percent morphology traits. Animals with AB genotype had significantly higher individual motility compared with AA genotype (P < 0.05). Also, animals with AA genotype had significantly the highest percent morphology compared with AB genotype (P < 0.1). The exon 3 of IGF-1 gene had significant effect on individual motility, concentration, morphology and water test traits. Animals with CT genotype had the highest sperm concentration (P < 0.1) and water test (P < 0.05) compared to CC and TT genotypes. Moreover, animals with TT genotype had significantly the highest percent morphology compared with other genotypes (P < 0.05). Briefly, the results indicated that individual motility, concentration, percent morphology and water test traits could be in association with IGF-1 genotypes. It might be concluded that polymorphisms in IGF-1gene can be considered to develop male fertility in future and for using in selection process of better animals under masker assisted selection programs. [ABSTRACT FROM AUTHOR]

This study analysed (i) the effect of human chorionic gonadotropin (hCG) and salmon gonadoliberine analogue (sGnRHa) on the effectiveness of induction of spermiation and (ii) the effect of latency time following the application of those spawning agents on the quantity and quality of the sperm of Eurasian perch, Perca fluviatilis , obtained during out-of-season spawning. For this study, pond-reared fish were used which had been acclimated to the controlled conditions. Three groups were distinguished which were treated with either saline (0.9% NaCl; control group), hCG (500 IU kg −1 ) or sGnRHa (100 μg kg −1 ). The fish were kept in a recirculating system at 12 °C throughout the study, during which sperm was collected every two days between the 2nd and 10th day following hormonal treatment. During the study, quantitative (e.g. sperm volume, total sperm production) and qualitative (measured with a computer-assisted sperm analysis system – i.e. CASA) parameters were monitored. The results of the study indicate that the hormonal treatment had a highly beneficial effect on the spermiation rate (100% in experimental groups from day 6 following injection) as well as quantity, which increased 50% in experimental groups (over 2200 × 10 9 of spermatozoa per kg of body weight) by day 4 following injection. For the sperm quality, both spawning agents tested had a rather positive effect, although sperm motility rate (MOT) was seen to be significantly reduced on day 10 following the application of hCG (MOT = 72.8% ± 8.1), which was not observed after the application of sGnRHa (minimum mean MOT 81.7% ± 6.1). The results clearly indicate that hormonal treatment had a positive effect on spermiation in Eurasian perch, most apparent from day 6 following injection, regardless of the hormonal agent used. Though application of sGnRHa allowed a high volume of high quality sperm to be stripped for two days longer (up to day 10 post-injection) compared to the application of hCG. [ABSTRACT FROM AUTHOR]

The aim of this study was to examine apoptotic markers in fresh and frozen-thawed feline spermatozoa collected via urethral catheterization and epididymal slicing. Caspase activation, DNA fragmentation, and phosphatidylserine externalization were evaluated using flow cytometry in sperm cells from both sources before and after cryopreservation. The study revealed no differences between urethral and epididymal spermatozoa, both in fresh and frozen-thawed samples. The level of apoptotic changes in sperm cells in fresh feline semen was low: 0.8 ± 0.8% of live urethral and 0.4 ± 0.4% of live epididymal spermatozoa showed active caspases; 1.6 ± 0.9% and 2.1 ± 1.9%, respectively, showed DNA fragmentation; and 0.3 ± 0.2% and 1.0 ± 1.3%, respectively, showed phosphatidylserine externalization. In both types of sperm cells, cryopreservation did not induce a significant increase in caspase activation (urethral: from 3.9 ± 3.2% to 7.5 ± 5.0%; epididymal: from 4.7 ± 2.9% to 11.7 ± 8.5%). In urethral spermatozoa, phosphatidylserine externalization in live cells was significantly (P < 0.05) increased after thawing (from 0.3 ± 0.2% to 2.7 ± 2.5%). This increase was not noted for epididymal spermatozoa (from 1.0 ± 1.3% to 1.7 ± 1.3%). No significant changes in DNA fragmentation were observed (2.1 ± 0.8% and 1.7 ± 1.0%). In conclusion, both urethral and epididymal feline spermatozoa showed equally low levels of apoptotic-like changes. Hence, apoptotic alterations seem to play only a minor role, if any, in urethral and epididymal feline spermatozoa. The deterioration of sperm quality after freezing and thawing is more likely connected with direct damage to the cells than to activation of apoptotic processes. [ABSTRACT FROM AUTHOR]

Fish tambaqui ( Colossoma macropomum ) is the native Brazilian fish with the highest agricultural production under intensive aquaculture in South America. However, the decrease in the genetic variability in fish farms has become necessary the improvement of cryopreservation process through new statistical studies of spermatozoa (like subpopulation studies). The evaluation of the kinetic data obtained with a computer-assisted sperm analysis system, applying a two-step cluster analysis, yielded in tambaqui three different subpopulations in fresh sperm: SP1, considered as a slow nonlinear subpopulation; SP2, considered as a fast nonlinear subpopulation, and finally; SP3, considered as a fast linear subpopulation. For cryopreserved sperm, the cluster analysis yielded only two sperm subpopulations: SP1′, considered as a slow nonlinear subpopulation and SP2′, which seemed to be an intermediate subpopulation (showing medium motility and velocity values) merged from SP2 and SP3 obtained from fresh sperm. Coefficients of correlation ( r ) and determination ( r 2 ) between the sperm subpopulations from fresh sperm and the fertilization rates were calculated, and SP2 and SP3 (the fast-spermatozoa subpopulations) showed a high-positive correlation with the fertilization rates ( r = 0.93 and 0.79, respectively). In addition, the positive significant correlations found in curvilinear velocity ( r = 0.78), straight line velocity ( r = 0.57), and average velocity ( r = 0.75) indicate that sperm kinetic features seem to be a key factor in the fertilization process in tambaqui, as occur in other fish species. [ABSTRACT FROM AUTHOR]

The first aim of this study was to provide a comprehensive analysis of structural and functional features of spermatozoa in semen collected from the same cat by two methods: urethral catheterization and epididymis slicing. The second aim was to assess if feline urethral (CT) and epididymal (EP) spermatozoa undergo the same changes during cryopreservation and to compare the postthaw characteristics of spermatozoa collected by the two methods. In the first phase, CT and EP semen were collected from 20 cats, and sperm motility, viability, morphology, computer-assisted sperm analysis (CASA) parameters, membrane and acrosome integrity, mitochondrial potential, lipid peroxidation, and chromatin status were assessed. In the second phase, both types of semen collected from 10 cats were cryopreserved, thawed, and the same sperm parameters were assessed as in fresh semen. Fresh CT spermatozoa (phase I) showed higher (P < 0.05) motility (subjective: median 75.0% vs. 62.5%; by CASA: mean ± SD 60.2 ± 10.7% vs. 43.1 ± 16.7%), morphology (mean ± SD, 57.5 ± 9.6% vs. 45.2 ± 15.9%), and membrane integrity (median live: 89.2% vs. 79.8%). Other parameters were not different between CT and EP spermatozoa. After cryopreservation (phase II), spermatozoa from both types of semen did not differ significantly, except for lipid peroxidation of live sperm cells (median CT: 3.5%, EP: 1.7%, P < 0.05). Urethral and EP spermatozoa showed a similar, significant drop in motility (CT to 18.6 ± 10.3% and EP to 21.6 ± 12.1%, P < 0.05), progressive motility (CT to 6.8 ± 5.9% and EP to 8.3 ± 8.8%, P < 0.05), and rapid movement (from 34.3 ± 20.6% to 8.5 ± 7.0% in CT and from 26.0 ± 14.7% to 10.1 ± 10.4% in EP, P < 0.05), whereas other motion characteristics assessed by CASA were not affected (P > 0.05). The strongest change after cryopreservation was noted in high mitochondrial potential (median CT: 1.3%, EP: 2.2%). Although cryopreservation increased acrosome damage and lipid peroxidation, the level of these changes in the population of live sperm cells remained low (median acrosome damage: CT: 3.3%, EP: 4.5%, lipid peroxidation CT: 3.5%, EP: 1.7%). Cryopreservation did not affect chromatin structure (median percent DNA fragmentation index, CT: 3.3%, EP: 2.3%). In this study, we confirmed that urethral catheterization for collection of semen allows the retrieval of spermatozoa with quality equally good as in those obtained by epididymal slicing. Spermatozoa from both types of semen collected showed similar characteristics after freezing/thawing so both types can be used for semen banking. [ABSTRACT FROM AUTHOR]

This study was designed to describe and compare basic semen characteristics and sperm motility parameters obtained via computer-assisted sperm analysis (CASA) in feline semen collected from the urethra and epididymis, on the basis of large, unselected population of domestic cats. The semen collected from 214 males was subjected for routine semen assessment and CASA evaluation. Semen collected by urethral catheterization (CT) and by epididymal slicing (EP) has comparable characteristics according to total sperm count (47.7 ± 42.1 and 52.9 ± 45.0), subjective motility (71.1 ± 17.0 and 69.3 ± 13.9), viability (74.9 ± 13.4 and 76.7 ± 10.6), and morphology (52.6 ± 19.0 and 47.2 ± 17.4). The study of a large feline population confirmed a high incidence of teratospermy in cats, which negatively affects sperm motility parameters assessed by CASA. A lack of a correlation between CT and EP semen for total sperm count and viability, as well as occasional gross differences between the morphology of CT and EP semen of the same cat suggests that many factors may affect sperm cells, and the fertility and/or infertility of patients should not be assessed after examining only one sample. Additionally, technical problems with assessment of EP samples (understated results) suggest that CT semen is more appropriate for an analysis by CASA than EP. [ABSTRACT FROM AUTHOR]

This study was conducted to investigate the effects of two carotenoids (astaxanthin and β-carotene) on the sperm quality of goldfish Carassius auratus (Linnaeus, 1758). For this purpose, six diets containing concentrations of 50, 100, and 150 mg/kg of synthetic astaxanthin and β-carotene were added to a basic carp diet. One group of fish was also fed with a control diet (no added carotenoids). Osmolality, spermatocrit value, and sperm concentration significantly increased in the treatment supplemented with 150 mg/kg of astaxanthin (296.6 ± 1.1 mOsm/kg; 29.2 ± 0.6%; 17.2 ± 0.4 × 10 9 cells/mL, respectively) and β-carotene (295.2 ± 2.1 mOsm/kg; 32.5 ± 1.6%; 17.9 ± 0.5 × 10 9 cells/mL, respectively). The highest concentration of astaxanthin (10.4 ± 1.4 mg/kg) was recorded in the treatment of A 150 (P < 0.05) and did not differ between β-carotene treatments. The highest motility was observed in the A 150 and B 150 treatments, and the lowest was observed in the control group (P < 0.05). The artificial fertilization of the treated males with the similar females (fed with the control diet) showed that the fertilization rate in the A 150 treatments was higher than in the other treatments (P < 0.05). In conclusion, dietary supplementation with 150 mg/kg of astaxanthin improves osmolality, motility, fertilization rate, and sperm concentration. [ABSTRACT FROM AUTHOR]

The aims of this study were to determine the sperm quality of frozen–thawed donkey sperm samples after single-layer centrifugation (SLC) using Androcoll-E in comparison to sperm washing or no centrifugation and to determine if the effect on the sperm quality after SLC or sperm washing depends on the quality of the sample. Frozen–thawed sperm samples from Andalusian donkeys were divided into three aliquots, and they were processed using three different techniques after thawing: uncentrifuged diluted control (UDC), sperm washing (SW), and SLC. Afterward, sperm quality index was estimated by integrating all parameters (total and progressive sperm motility, membrane integrity, and DNA fragmentation) in a single value. The relationship between the sperm quality of thawed UDC samples and the effect on sperm parameters in SW and SLC-selected samples was assessed. Sperm quality index was significantly higher (P < 0.001) in SLC (0.8 ± 0.0) samples than that in UDC (0.6 ± 0.0) and SW (0.6 ± 0.0) samples, regardless of the sperm quality index after thawing of the sperm sample. In conclusion, SLC of frozen–thawed donkey spermatozoa using Androcoll-E-Small can be a suitable procedure for selecting frozen–thawed donkey sperm with better quality, in particular in those samples where an improvement in motility is needed. [ABSTRACT FROM AUTHOR]

The main aim of this study was to determine whether acrosin activity could predict boar sperm freezability. For this purpose, we characterized the changes in sperm quality and acrosin activity throughout the cryopreservation procedure of sperm samples from 30 Pietrain boars by analyzing four critical steps: step 1 (extended sperm at 15 °C), step 2 (cooled sperm at 5 °C), step 3 (30 minutes postthaw), and step 4 (240 minutes postthaw). Freezability ejaculate groups were set on the basis of sperm motility and membrane integrity after freeze–thawing. Results obtained highlighted the low predictive value in terms of freezability of sperm motility and kinematics and sperm membrane integrity, as no differences between good and poor freezability ejaculates were seen before cryopreservation. Significant differences (P < 0.05) between ejaculate groups were observed in the cooling step at 5 °C for sperm kinetic parameters, and after thawing for sperm motility and membrane integrity. In contrast, acrosin activity appeared as an indicator of boar sperm freezability because the differences (P < 0.05) between good and poor freezability ejaculates manifested yet in extended samples at 15 °C. On the other hand, we also found that variations in sperm kinematics, membrane lipid disorder, intracellular calcium content, acrosome integrity, and acrosin activity throughout the cryopreservation procedure were indicative of a significant damage in spermatozoa during the cooling step in both ejaculate groups. In conclusion, the main finding of our study is that acrosin activity can be used as a reliable predictor of boar sperm freezability because it differs significantly between good and poor freezability ejaculates yet before freeze–thawing procedures took place, i.e., in the refrigeration step at 15 °C. [ABSTRACT FROM AUTHOR]

The present approach was designed to evaluate the extender effects on sperm quality and fertility of short-term refrigerated seminal doses from Landrace boars lodged in husbandry-controlled conditions. For this purpose, we analyzed the sperm quality of seminal doses diluted in short-term (Beltsville Thawing Solution) and extra-long-term (Duragen) extenders from Days 0 to 2 of storage at 17 °C during an 8-month period. Pregnancy rates and litter size were evaluated from double inseminations within an interval of 12 hours (36 and 48 hours of refrigeration) of multiparous females using seminal doses diluted in each extender type. Sperm quality was assessed from the analyses of sperm motility and kinetics, sperm viability, expressed as plasma and acrosome membrane integrity, membrane lipid disorder, intracellular calcium levels, and acrosin activity. Results indicated significant differences between the extenders in the sperm quality of seminal doses. Therefore, the seminal doses diluted in Duragen had higher percentages of progressive motile spermatozoa and membrane-intact spermatozoa than those diluted in Beltsville Thawing Solution throughout all the experimental months. Nevertheless, despite these differences in preserving the sperm quality, pregnancy rates (>90%) and litter sizes (>10 piglets born per litter) were similar between the extenders. Our results had great relevance from a practical point of view because they reported lack of an extender effect on the reproductive performance of seminal doses during short-tem storage. [ABSTRACT FROM AUTHOR]

This study examines the viability of Mycoplasma agalactiae (Ma) and Mycoplasma mycoides subsp capri (Mmc) during 150 minutes of incubation at 37 °C in contaminated diluted semen (DS) doses. The effects of the presence of both microorganisms on sperm viability, motility, and morphology were also examined. In a second experiment, the viability of Ma and its effects on sperm viability were determined in ejaculate samples and skimmed milk semen extender samples. Ma and Mmc were able to survive in DS at concentrations considered infectious, and no significant differences in mean concentrations were detected (7.1 log colony-forming units [CFU]/mL). However, initial concentration of Ma declined (P < 0.05) from 7.5 to 6.9 log CFU/mL and Mmc declined (P < 0.05) from 7.7 to 7.1 log CFU/mL after incubation. Conversely, ejaculate concentrations of Ma increased significantly (from 7.1 to 7.4 log CFU/mL, P < 0.05). These observations suggest that the natural breeding medium is more suitable for Ma than the medium used for artificial insemination (AI). The presence of Mmc slightly reduced sperm viability in the DS (from 21.7% to 16.6%, P < 0.05). The absence of major effects on sperm quality could lead to the unnoticed use of semen contaminated with Ma and Mmc for AI. As both bacteria were able to survive the conditions of ejaculates and semen doses, these findings suggest a risk of venereal transmission of contagious agalactia and support the use of mycoplasma-free semen samples for (AI). [ABSTRACT FROM AUTHOR]

The aims of this study were to evaluate the characteristics of the reproductive classes and semen quality in curimbatá ( Prochilodus lineatus ) breeders maintained in two different rearing systems. To achieve this goal, cages (Cs) and earthen ponds (EPs) were used as experimental systems to provide unsuitable and suitable conditions, respectively. The fish were maintained under the experimental conditions for 18 months. During this period, males were randomly sampled every 2 months for biometric analysis (n = 30 per sample) and for an evaluation of selected characteristics of the testes (n = 5 per sample). After this period, males maintained in EPs and males maintained in Cs (CMs) were evaluated in induced breeding experiments. We observed that rearing P. lineatus in a C at a high stocking density for the long 18-month period of study produced reductions in growth, testis development, gonadosomatic index values, and sperm quality in the fish. We found differences between the groups in all the reproductive classes examined, especially in the regression class, which showed a pronounced accumulation of immature germ cells in the CMs. In this group, we also noted a less intense transition from a continuous to discontinuous germinal epithelium, with an extended and abnormal but less intense spermatogenic period resulting in decreases in semen volume and sperm concentration in the breeding season. Together, such dysfunctions resulted in the production of low-quality sperm in the CMs, as demonstrated by lower-quality DNA (as evaluated by the comet assay), low fertilization success, and low hatching success. In conclusion, to ensure high-quality semen in P. lineatus , appropriate management conditions must be provided throughout the reproductive cycle, especially for the regressed class, even in winter, two seasons before the breeding season. [ABSTRACT FROM AUTHOR]

Canine epididymal spermatozoa have a low freeze-tolerance ability compared with ejaculated spermatozoa, which could arise from the absence of prostatic fluid (PF). Therefore, the purpose of this work was to elucidate the influence of PF on the quality of canine epididymal sperm before and after freezing. Caudae epididymides were retrieved from eight dogs after routine castration. Spermatozoa were released by slicing the tissue and were extended in either Tris solution or PF before freezing. Frozen sperm samples were thawed at 70 °C for 8 seconds in a waterbath. Sperm concentration, motility using computer-assisted sperm analysis, morphology, plasma membrane, acrosome and chromatin integrity were assessed in the fresh sperm samples (after 20 minutes incubation) and at 0 and 4 hours after thawing. Progressive motility, distance straight line, distance average path, average path velocity, curvilinear velocity, straight line velocity, straightness, linearity, wobble, and beat cross frequency were significantly increased after extraction into PF. There was a higher proportion of spermatozoa with DNA damage in the PF treatment group at 4 hours after thawing than in the Tris treatment group (15.8% vs. 6.7%, P < 0.05). These results suggest that the addition of PF to canine spermatozoa activates sperm motility in fresh spermatozoa but has a negative effect on chromatin integrity after freezing–thawing. [ABSTRACT FROM AUTHOR]

Abstract: Many attempts have been made to identify laboratory tests that are predictive of sperm fertility, both to improve the quality of stallion semen doses for artificial insemination (AI) and to identify potential breeding sires if no fertility data are available. Sperm quality at the stud is mostly evaluated by assessing subjective motility, although this parameter can be poorly indicative of fertility. Sperm morphology and chromatin integrity in Swedish stallions are correlated to pregnancy rate after AI. Because single layer centrifugation (SLC) selects for spermatozoa with normal morphology and good chromatin, retrospective analysis was carried out to investigate whether sperm yield after SLC is linked to potential fertility. Commercial semen doses for AI from 24 stallions (five stallions with four ejaculates each, 19 stallions with three ejaculates each; n = 77) obtained during the breeding season were cooled, and sent overnight to the Swedish University of Agricultural Sciences in an insulated box for evaluation, with other doses being sent to studs for commercial AI. On arrival at Swedish University of Agricultural Sciences, the semen was used for SLC and also for evaluation of sperm motility, membrane integrity, chromatin integrity, and morphology. The seasonal pregnancy rates for each stallion were available. The yield of progressively motile spermatozoa after SLC (calculated as a proportion of the initial load) was found to be highly correlated with pregnancy rate (r = 0.75; P < 0.001). Chromatin damage was highly negatively correlated with pregnancy rate (r = −0.69; P < 0.001). Pregnancy rate was also correlated with membrane integrity (r = 0.58; P < 0.01), progressive motility (r = 0.63; P < 0.01), and normal morphology (r = 0.45; P < 0.05). In conclusion, these preliminary results show that sperm yield after SLC is related to the potential fertility of the original ejaculate, and could be an alternative indicator of stallion fertility if breeding data are not available. Single layer centrifugation is fast (30 minutes) and does not require expensive equipment, whereas other assays require a flow cytometer and/or specialist skills. An additional option could be to transport semen doses to a laboratory for SLC if the stud personnel do not want to perform the procedure themselves. [Copyright &y& Elsevier]

Abstract: The aim of this study was to investigate whether single-layer centrifugation (SLC) with Androcoll-C could select good quality spermatozoa, including those with specific motility patterns, from doses of frozen dog semen. Semen from five dogs was collected and cryopreserved following a standard protocol. After thawing, the semen samples were divided in two aliquots, one of which was used as a control and the other one processed by SLC. Assessment of sperm motility (assessed by computer-assisted semen analysis), morphology (Diff-Quick staining), viability (dual staining with propidium iodine/acridine orange), and acrosome integrity (dual staining with propidium iodine/isothiocyanate-labeled peanut [Arachis hypogaea] agglutinin) were performed on aliquots of fresh semen, frozen-thawed control samples, and frozen-thawed SLC-treated preparations. A multivariate clustering procedure separated 57,577 motile spermatozoa into three subpopulations (sP): sP1 consisted of poorly active and nonprogressive spermatozoa (48.8%), sP2 consisted of moderately slow but progressive spermatozoa (13.3%), and sP3 consisted of highly active and/or progressive spermatozoa (37.8%). SLC with Androcoll-C yielded sperm suspensions with improved motility, viability, and acrosome integrity (P < 0.01). The frozen-thawed SLC-treated samples were enriched in sP3, representing 38.5% of the sperm population. Likewise, sP2 was more frequently observed after SLC, but not significantly so. From these results, we concluded that for dog semen samples selected by SLC with Androcoll-C after thawing, the sperm quality parameters, including motility patterns, are better than in frozen-thawed control samples. [Copyright &y& Elsevier]