Study of Sperm Concentration,Seminal Plasma Composition and their Physiological Correlation in the Persian Sturgeon,Acipenser persicus

The objectives of the present study were to determine ionic and organic composition of seminal plasma, sperm concentration and their relationships in the Persian sturgeon (Acipenser persicus). In this regard, ionic content (Na⁺, K⁺, Cl^?, Ca²⁺ and Mg²⁺) and organic content (total protein, glucose, cholesterol and triglyceride) along with sperm concentration were measured in 17 specimens of the Persian sturgeon. The seminal plasma contained 59.53 ± 2.56 mm /l sodium, 9.1 ± 1.42 mm chloride, 4.72 ± 0.3 mm potassium, 1.45 ± 0.075 mm calcium and 0.7 ± 0.072 mm magnesium. The following organic contents were found: total protein 0.11 ± 0.02 g/dl, glucose 22.18 ± 4.16 mg/dl, cholesterol 6.67 ± 1.04 mg/dl and triglyceride 15.2 ± 0.65 mg/dl. The mean sperm concentration was estimated to be 1.6 ± 0.12 (×10⁹ sperm/ml). A significant relationship was found between sperm concentration and K⁺ of seminal plasma (r = 0.533, p < 0.05). Significant correlations were observed between ionic contents: Na⁺ vs Cl^? (r = ?0.854, p < 0.01) and Mg²⁺ vs K⁺ (?0.583, p < 0.05). Also, level of triglyceride was negatively correlated with Mg²⁺ (r = ?0.503, p < 0.05). Presented data could be considered as a complementary study for developing special extenders and protectant solutions for improving artificial fertilization in this valuable species.     

Assessment of Sperm Function Parameters and DNA Fragmentation in Ejaculated Alpaca Sperm (Lama Pacos) by Flow Cytometry

Flow cytometry has been shown to be an accurate and highly reproducible tool for the analysis of sperm function. The main objective of this study was to assess sperm function parameters in ejaculated alpaca sperm by flow cytometry. Semen samples were collected from six alpaca males and processed for flow cytometric analysis of sperm viability and plasma membrane integrity using SYBR‐14?PI staining; acrosomal membrane integrity using FITC‐conjugated Pisum Sativum Agglutinin?PI labelling; mitochondrial membrane potential (Δψm) by staining with JC‐1 and DNA Fragmentation Index (DFI) by TUNEL. The results indicate that the mean value for sperm viability was 57 ± 8 %. Spermatozoa with intact acrosome membrane was 87.9 ± 5%, and viable sperm with intact acrosomal membrane was 46.8 ± 9%, high mitochondrial membrane potential (Δψm) was detected in 66.32 ± 9.51% of spermatozoa and mean DFI value was 0.91 ± 0.9%. The DFI was inversely correlated with high Δψm (p = 0.04; r = ?0.41) and with plasma membrane integrity (p = 0.01; r = ?0.47). To our knowledge, this is the first report of the assessment on the same sample of several parameters of sperm function in ejaculated alpaca sperm by flow cytometry.     

The Control of Reactive Oxygen Species Influences Porcine Oocyte In Vitro Maturation

The aim of this study was to examine the effect of varying intracellular reactive oxygen species (ROS) levels during oocyte in vitro maturation with enzymatic ROS production systems (xanthine + xanthine oxidase or xanthine + xanthine oxidase + catalase), scavenger systems (catalase or superoxide dismutase + catalase) or cysteine on porcine oocyte maturation. Oocyte ROS levels showed an increase when H₂O₂ or O₂?^‐ production systems were added to the culture medium (p < 0.05). On the other hand, the presence of ROS scavengers in the maturation medium did not modify oocyte ROS levels compared with the control after 48 h of maturation, but the addition of cysteine induced a decrease in oocyte ROS levels (p < 0.05). The ROS production systems used in this work did not modified the percentage of oocyte nuclear maturation, but increased the decondensation of sperm head (p < 0.05) and decreased the pronuclear formation (p < 0.05). In turn, the addition of O₂?^‐ and H₂O₂ scavenging systems during in vitro maturation did not modify the percentage of oocytes reaching metaphase II nor the oocytes with decondensed sperm head or pronuclei after fertilization. However, both parameters increased in the presence of cysteine (p < 0.05). The exogenous generation of O₂?^‐ and H₂O₂ during oocyte in vitro maturation would not affect nuclear maturation or later sperm penetration, but most of the spermatozoa cannot progress to form the pronuclei after fusion with the oocyte. The decrease in endogenous ROS levels by the addition of cysteine would improve pronuclear formation after sperm penetration.     

Impact of 24-h Cooling Prior to Freezing on the Survival of Domestic Cat (Felis catus) Epididymal Sperm

The aim of this study was to investigate the impact of a 24-h cooling period prior to freezing on domestic cat epididymal sperm viability. Fifteen tomcats were submitted to routine orchiectomy and sperm samples were retrieved from both epididymides in a Tris–glucose–20% egg yolk extender. For each tomcat, the diluted sperm was split into two equal volumes and cooled to 5°C at a rate of 0.5°C/min; one sample for 60 min (control) and the other for 24 h (cooled). After the cooling period, samples from both groups were frozen using an identical freezing protocol. Sperm samples were evaluated in three different periods: immediately after harvesting, after cooling at 5°C for 24 h (cooled group) and after freezing–thawing of control and cooled groups. Evaluations consisted of sperm motility and progressive status, sperm morphology and plasma membrane integrity (PMI) using two fluorescent probes. After cooling for 24 h, a decrease (p < 0.05) in sperm motility, progressive status and PMI was observed when compared to sperm samples immediately after collection. Comparing the results obtained after thawing, no difference (p < 0.05) was found regarding sperm motility, progressive status, PMI and sperm morphology between control and cooled groups. The results from the present study show that cooling cat epididymal spermatozoa at 5°C for 24 h prior to freezing does not lead to major damage of spermatozoa impairing the freeze–thaw process.     

Improvement in In Vitro Fertilization Rate,Decrease in Reactive Oxygen Species and Spermatozoa Death Incidence in Rams by Dietary Fish Oil

Our aim was to evaluate the effects of fish oil feeding on sperm classical parameters, level of reactive oxygen spices (ROS), spermatozoa death incidence and in vitro fertilization (IVF) rate in rams. We randomly assigned nine rams, into two experimental groups (isoenergetic and isonitrogenous rations with constant level of vitamin E supplement): control (CTR; n = 5) and fish oil (FO; n = 4, 35 g/day/ram). Diets were fed for 70 days during the physiological breeding season. After a 21‐day dietary adaptation period, semen was collected weekly from each ram by an artificial vagina. Sperm classical parameters were determined by the computer‐assisted sperm analyzer system (CASA), and it was prepared for IVF process by swim‐up technique. These evaluations were performed during the first and last weeks of sampling. Intracellular ROS level and spermatozoa death incidence were detected by flow cytometry on a weekly basis after adaptation. Data were analysed with SPSS 15. The volume, concentration (3.6 and 2.7 × 10⁹/ml) and sperm progressive motility (60 and 48%) were significantly improved in the FO group compared with the CTR (p < 0.05). A comparison of two‐cell stage embryos following IVF in the two groups showed a significantly higher fertilization rate in the FO group (56%) compared with the CTR (49%). Superoxide anion (O₂^?) rate was significantly lower (p < 0.05) at the third week of sampling in the FO. Although the H₂O₂ rate was numerically lower in the FO group compared with the CTR, this difference was not significant. In addition, apoptosis showed a significant difference in the third week of sampling (15 and 30% for FO and CTR, respectively; p < 0.05). Overall, adding fish oil to the ram diet not only improved sperm quality and IVF results, it also could reduce oxygen‐free radicals and the incidence of spermatozoa death.     

Effect of Progesterone and Ionomycin on Domestic Cat Sperm Motility Patterns and Acrosome Reaction

This study was aimed at assessing the changes in sperm motion patterns and the percentage of acrosome reaction (AR) in domestic cat semen after treatment with either ionomycin or progesterone (P₄). Ten ejaculates were collected from five tomcats using an artificial vagina, and were diluted, centrifuged and resuspended in a capacitation medium. Samples were evaluated and divided into seven equal aliquots and, after 2 h at 25°C, were incubated for 30 min at 38°C in 5% CO₂ and then analyzed. Computer-assisted sperm analysis and a combination of three fluorescent probes were used to assess sperm plasma, acrosomal membrane integrity and mitochondrial transmembrane potential. Thirty minutes after the start of incubation, P₄ was added (10 μg/ml) to the P1 group. Groups P2 and P3 were supplemented with P₄ (10 and 20 μg/ml, respectively) only after 2 h of incubation, and groups I1 and I2 were supplemented with ionomycin (4 and 8 μ m , respectively) 2 h after incubation. Group E was supplemented with ethanol (0.6%) at 2 h after incubation and group C received no supplementation. Ionomycin and P₄ treatments led to a hyperactivation-like sperm motion and an increase (p < 0.05) in the percentage of AR. Although a higher (p < 0.05) percentage of AR was obtained in group I2 when compared with all P₄ groups, a decrease (p < 0.05) in total and progressive motility was observed in I2 group. As I1 group was similar to I2 to induce AR without diminishing sperm motility, we can conclude that ionomycin at 4 μ m seems to be more suitable to trigger AR in domestic cat sperm.     

Retracted: Effects of multiple collections on spermatozoa quality of Persian sturgeon,Acipenser persicus: motility,density and seminal plasma composition

In this study, we investigated the effects of multiple collections of sperm on the endangered Persian sturgeon, Acipenser persicus, in terms of a number of sperm functional parameters (percentage of motile spermatozoa, total time period of motility and sperm concentration) as well as on the ionic composition, protein concentration and osmolality of seminal plasma. Semen samples were collected from 12 induced male fish in three experimental groups that had been injected intramuscularly with LHRH‐A₂, at dosages of 5 μg/kg body weight, at a number of time regimes: at 12 h, 17 h and 24 h after spawning induction (1); at 24, 29 and 34 h after spawning induction (2); and at 36, 41 and 46 h after spawning induction (3). The percentage of motile spermatozoa and the period of sperm motility decreased significantly (p < 0.05) after the second and third collections. The concentration of spermatozoa decreased after the third collection, but this decline was not significant. No significant effect of multiple collections on protein concentration and ionic content (with exception of the Cl^? ion) of seminal plasma was observed. In all experimental groups, a moderate impact of sequential collection on the osmolality (p < 0.05) of seminal plasma was observed. This study provides new data on the effects of multiple collections on spermatological characteristics in the Persian sturgeon. Our results confirm that sequential stripping after the third collections has a negative effect on a number of functional parameters associated with sperm.     

Morphology and Chromatin Integrity of Stallion Spermatozoa Prepared by Density Gradient and Single Layer Centrifugation Through Silica Colloids

The objective was to investigate whether it is possible to improve the quality of stallion semen, with respect to sperm morphology and chromatin integrity, both of which have been linked to fertility, using either density gradient centrifugation (DGC) or a new method, hereby named single layer centrifugation (SLC). The two methods of colloidal centrifugation were evaluated using 38 ejaculates from 10 stallions. Sperm morphology, subjective motility and sperm chromatin integrity were compared in uncentrifuged samples and in centrifuged sperm preparations. The proportion of morphologically normal spermatozoa varied between stallions (p < 0.001) and was increased by both methods of colloidal centrifugation (median value before centrifugation 67.5%; after SLC 78%; after DGC 77%; p < 0.001). The incidence of certain abnormalities was reduced, e.g. proximal cytoplasmic droplets were reduced from 12.9% to 8.8% (p < 0.001), and mid-piece defects from 5.3% to 1.4% (p < 0.05). Similarly, sperm motility and chromatin integrity were significantly improved (p < 0.001), with no difference between the two centrifugation methods. Centrifugation through colloids can enrich the proportions of stallion spermatozoa with normal morphology and normal chromatin structure in sperm preparations. The new method, SLC, was as effective as DGC in selecting motile stallion spermatozoa with normal morphology and intact chromatin. SLC, being simpler to use than DGC, would be appropriate for routine use by stud personnel to improve stallion sperm quality in insemination doses.     

Lactoferrin increases sperm membrane functionality of frozen equine semen

During cryopreservation, sperm was submitted to an increase in reactive oxygen species generation. This work aimed to improve the quality of frozen equine sperm after the addition of antioxidants lactoferrin (Lf) and catalase (Cat) to a freezing extender. Semen from six stallions was frozen with the extenders: F1) control, INRA 82 freezing extender, F2) F1 + 500 μg/ml Lf and F3) F1 + 200 IU/ml Cat. After thawing, sperm motility parameters, membrane functionality and integrity, and acrosome integrity and spontaneous acrosome‐reacted sperm were evaluated with a computer‐assisted sperm analysis, a hypoosmotic swelling test and epifluorescent microscopy, respectively. Nitrite, hydroperoxide and iron concentrations of frozen semen were measured with spectrophotometry. The percentage of functional membrane sperm treated with Lf was higher (50.7% ± 11.6%) compared to that of the control (37.6% ± 15.6%), while the iron (61.4 ± 11.6 vs 73.3 ± 13.8 mg/dl) and nitrite concentrations (16.3 ± 7.1 vs 25.9 ± 4.2 μM/μg protein) were lower, respectively (p < .05). Thus, it can be suggested that Lf protect stallion spermatozoon during freezing as it has increased the percentage of sperm with functional membrane and decreased the lipid oxidant agents.     

Effects of Straw Volume and Equex-STM® on Boar Sperm Quality after Cryopreservation

The present experiments were designed to study the effect of adding the detergent Equex-STM^® to freezing extender, and of straw volume (0.25 ml vs 0.5 ml), on boar sperm quality after cryopreservation. Three ejaculates from each of four purebred boars (three Landrace and one Yorkshire) were collected and frozen with a lactose-egg yolk extender containing glycerol with or without 1.5% Equex-STM^®. The extended semen was loaded into either 0.25- or 0.5-ml straws. The straws were placed in liquid nitrogen (LN₂) vapour approximately 3 cm above the level of LN₂ for 20 min and then were plunged into LN₂. Thawing was achieved in warm water at 50°C for 12 s and then was incubated in a 38°C water-bath for 30 min before evaluating sperm quality. Results showed that the individual motility, viability and acrosomal normal apical ridge (NAR) were improved (p < 0.001) when Equex-STM^® was added to the freezing extender. There was no difference (p   =   0.48) in sperm motility between 0.25- and 0.5-ml straws when Equex-STM^® was added. The percentages of viable and of NAR sperm in 0.5-ml straws were higher than those in 0.25-ml straws (p   =   0.02, p   =   0.0003 respectively). The percentages of membrane intact sperm evaluated using the short hypo-osmotic swelling test were not affected by straw volume or the adding of Equex-STM^® (p   >   0.05). The results of these investigations suggested that Equex-STM^® exerts a beneficial effect on the quality of cryopreserved boar semen and this cryopreservation protocol was favourable for a 0.5-ml straw.     

辅酶Q10对绒山羊精液冷冻保存效果的影响

旨在探讨辅酶Q10对绒山羊精液冷冻保存效果的影响。利用添加不同浓度辅酶Q10(4、40、400?滋g/mL)的精液冷冻稀释液对绒山羊精液样本进行冷冻保存,待冷冻精液解冻后,采用流式细胞仪和计算机辅助精液分析系统(CASAS)分别检测不同精液样本的精子活率、质膜完整率、顶体完整率、DNA完整率、线粒体膜电位和细胞内ROS水平。结果表明,当冷冻稀释液中添加浓度为40μg/mL辅酶Q10时,经历冷冻—解冻过程的绒山羊精液样本的精子活率、质膜完整率、顶体完整率均显著高于对照组(P<0.05);在冷冻稀释液中添加浓度为40μg/mL或400μg/mL的辅酶Q10均能显著提高线粒体膜电位并降低细胞内ROS水平(P<0.05)。综上所述,在冷冻稀释液中添加40μg/mL的辅酶Q10能够显著提高绒山羊精子抗氧化能力和冷冻保存效果。     

Effect of Antibiotics in Extender on Bacterial and Spermatozoal Quality of Cooled Buffalo (Bubalus bubalis) Bull Semen

The present study was designed to study the effect of traditional antibiotic combination (streptomycin and penicillin; SP) and relatively modern combination of antibiotics (gentamycin, tylosin, lincomycin and spectinomycin; GTLS) in extender on bacterial control and spermatozoal quality of liquid buffalo bull semen stored at 5°C. Semen collected from Nili‐Ravi buffalo bulls (n = 10) was diluted with skim milk extender containing either SP (streptomycin 1000 μg/ml and penicillin 1000 IU/ml), GTLS (gentamycin 500 μg/ml, tylosin 100 μg/ml, lincomycin 300 μg/ml and spectinomycin 600 μg/ml) or negative control with no antibiotics (NA). Liquid semen was stored at 5°C for 5 days. Aerobic bacteria isolated from buffalo semen were Pseudomonas aeruginosa and Staphylococcus aureus. The only facultative anaerobic bacterium isolated was Klebsiella pneumoniae. In vitro antibiotic sensitivity test revealed that Ps. aeruginosa and Staph. aureus were susceptible to gentamycin. Staphylococcus aureus and K. pneumoniae were susceptible to tylosin and linco‐spectinomycin. Total aerobic bacterial count was significantly lower in semen samples treated with GTLS than those of SP on third and fifth day of storage at 5°C. There was no difference (p > 0.05) in sperm motility, longevity and plasma membrane integrity (PMI) in extender containing SP or GTLS combination until the third day of storage at 5°C. On fifth day of storage sperm motility, longevity and PMI was significantly better in extender containing SP compared with GTLS and NA. Intact acrosomes, and sperm head, mid piece and tail abnormalities remained similar (p > 0.05) because of antibiotics up to 5 days of storage. In conclusion, GTLS is more capable than SP for bacterial control of buffalo bull semen. Moreover, GTLS and SP are equally efficient in preserving spermatozoal quality of extended buffalo bull semen for 3 days at 5°C.     

Use of Glutamine and Low Density Lipoproteins Isolated from Egg Yolk to Improve Buck Semen Freezing

To improve the results obtained with a reference cryopreservation extender (control extender: Triladyl® + 20% (v/v) egg yolk + 6.4% (v/v) glycerol) for freezing caprine semen, glutamine was added to 18 split ejaculates at concentrations of 0, 20, 40, 80 and 120 m m (experiment 1). In experiment 2, glutamine was added to 18 split ejaculates at concentrations of 20, 25, 30, 35 and 40 m m . In the third experiment, the egg yolk was replaced with the low-density lipoprotein (LDL) fraction of egg yolk. The quality of frozen then thawed spermatozoa in each extender was compared using computer-assisted semen analysis. In experiment 1, glutamine at concentrations of 20 m m and 40 m m significantly improved sperm motility compared with the control extender. However, at 120 m m , a significant decrease in motility and velocity was observed. In experiment 2, motility, curvilinear velocity and amplitude of lateral head displacement (ALH) were improved in glutamine at 25 m m compared with the control. In experiment 3, 8% LDL and 25 m m glutamine significantly improved sperm motility, straight line velocity and ALH. In the fourth experiment, the quality of the previously defined freezing extender (Triladyl® + 8% (v/v) LDL + 25 m m glutamine + 6.4% (v/v) glycerol) was tested by comparing acrosome, tail membrane, plasma membrane and DNA integrity in 18 split ejaculates of frozen then thawed spermatozoa with spermatozoa that had been frozen then thawed in the control extender, and with spermatozoa from fresh, unfrozen sperm. The percentage of spermatozoa with intact acrosomes and tail membranes was significantly higher with the newly defined extender than that observed with the control extender. There was no significant difference in the percentage of spermatozoa with intact DNA between the frozen and fresh semen.     

Arachidic Acid in Extender Improves Post‐thaw Parameters of Cryopreserved Nili‐Ravi Buffalo Bull Semen

Cryopreservation process reduces lipids and phospholipids from buffalo bull spermatozoa. It was therefore hypothesized that supplementation of fatty acid to extender may improve the post‐thaw quality of buffalo semen. The objective was to evaluate the effect of arachidic acid supplementation in extender on post‐thaw quality of buffalo bull (Bubalus bubalis) spermatozoa. Semen was collected from three adult Nili‐Ravi buffalo bulls of similar age group with artificial vagina (42°C) for 3 weeks (replicate). Qualified semen ejaculates (n = 18) were split into four aliquots and diluted in tris–citric acid extender containing 0.0 (control), 5.0, 10.0 and 20.0 ng/ml at 37°C having approximately 50 × 10⁶ spermatozoa/ml. Diluted semen was cooled to 4°C in 2 h and equilibrated for 4 h at 4°C. Cooled semen was filled in 0.5‐ml straws at 4°C, kept on liquid nitrogen vapours for 10 min and plunged in liquid nitrogen for storage. Thawing of frozen semen was performed after 24 h at 37°C for 30 s. Sperm progressive motility (%) was improved in a dose‐dependent manner by supplementing arachidic acid at 5.0, 10.0 and 20.0 ng/ml compared with control. Structural and functional integrity of sperm plasma membrane (%), number of acrosome‐intact live sperm (%) and sperm chromatin integrity (%) were better (p < 0.05) in extender having 5.0 ng/ml of arachidic acid compared with control. At 10.0 ng/ml, these values did not vary (p > 0.05) from those at 5.0 ng/ml. Further improvement in structural and functional integrity of sperm plasma membrane, number of acrosome‐intact live sperm and chromatin integrity was observed at 20.0 ng/ml of arachidic acid in extender. In conclusion, arachidic acid supplementation in extender improved the post‐thaw quality parameters of cryopreserved Nili‐Ravi buffalo bull spermatozoa. Among the arachidic acid concentrations studied, maximum improvement in post‐thaw semen quality parameters was observed at 20.0 ng/ml.     

Assessment of Fertility after Using Different Procedures to Thaw Ram Spermatozoa Frozen in Mini Straws

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In this study, fertility rates were compared after using different procedures (50°C and 70°C) to thaw ram spermatozoa frozen in mini straws. Semen from three, 1.5–2.5-year-old rams of the same breed, selected for use in an AI programme, was collected using an artificial vagina. The semen was diluted with a skim milk extender containing 7% glycerol (v/v), packed in 0.25-ml mini straws and frozen in a programmable freezer. Post-thaw sperm motility was assessed subjectively using a phase contrast microscope. Sperm membrane integrity was assessed with fluorescent dyes (Calcein AM/EthD-1). Statistically significant variation in the incidence of membrane integrity was found, both between rams and between freezing operations. Significant differences between the different thawing procedures used in this study were seen for membrane integrity (p < 0.01), as assessed with the fluorescent dyes (Calcein AM/EthD-1), but not for the post-thaw motility. The average fertility in this study was 39.7%, with a wide variation between freezing operations (not significant), rams (p < 0.001; 30.4, 33.3 and 64.6%) and flocks (p < 0.001, range: 14.8–61.6%). No statistically significant differences were found for the different thawing procedures, in terms of the fertility (39.0 and 40.4%, respectively) and the litter size (1.32 and 1.41, respectively). Thawing at 50°C for 9 s, instead of 70°C for 5 s, does not seem to further affect either fertility or litter size. The use of this lower temperature would facilitate the practical use of frozen–thawed ram semen under farm conditions in Sweden.     

Effect on Rendement Napole genotype on metabolic markers in Ossabaw pigs fed different levels of fat

We investigated effects of Rendement Napole (RN ) genotype on metabolic markers in Ossabaw pigs fed diets with different levels of dietary fat. Thirty‐two pigs, belonging to either the wild‐type (WT , rn⁺/rn⁺) or carrier (CAR , RN ^?/rn⁺) genotypes (n  = 16/genotype), were divided into two dietary groups, (high fat [HF ] or low fat [LF ]) diets, for 12 weeks (n  = 8 pigs/genotype/diet) after which pigs were killed for gene expression analysis by RT ‐PCR . Feeding HF diet caused increased daily gain (ADG , p  <  .05) and final body weight (BW ) (p  <  .05) in comparison with the LF diet (p  <  .05). Feed efficiency (gain:feed) was higher (p  <  .05) in pigs on the HF and was higher (p  <  .05) in CAR pigs compared to WT . There was genotype × diet interaction (p  =  .05) on final BW such that CAR animals on LF diet had the same final BW as animals of both genotypes on HF diet. Carrier pigs on LF diet had higher (p  <  .05) average daily gain and gain:feed than WT pigs. There was a trend (p  <  .08) for a higher feed consumption in pigs on the LF diet. Backfat thickness was higher (p  <  .01) in pigs on the HF diet. Serum triglyceride was higher (0.62 vs. 0.33 mg/dl, p  <  .01) in pigs on HF diet. Serum insulin was higher (p  <  .05) in CAR versus WT pigs (0.40 vs. 0.015 μg/ml). Pigs on the HF diet had a higher (p  <  .05) serum insulin compared to those on the LF diet (0.032 vs. 0.023 μg/ml). Carnitine palmitoyl transferase 1‐alpha was higher (p  <  .05) in the longissimus dorsi and semitendinosus muscles of pigs on HF diet. Acyl‐CoA oxidase I was elevated (p  <  .05) in the liver of pigs on HF diet. Fatty acid synthase was lower in the longissimus dorsi muscle, liver and mesenteric fat (p  <  .05) of carrier pigs. The RN gene regulates specific metabolic markers in the Ossabaw pigs.     

Sperm preparation through Sephadex™ filtration improves in vitro fertilization rate of buffalo oocytes

Routinely, swim‐up method is used to separate high‐quality sperm; however, long processing time and close cell‐to‐cell contact during the centrifugation step are inevitable elements of oxidative stress to sperm. The objective was to evaluate Sephadex^? and glass wool filtration to separate motile, intact and viable sperm for in vitro fertilization in buffalo. The cumulus–oocyte complexes (COC s) were collected from ovaries of slaughtered buffaloes by aspiration and matured for 24 hr in CO ₂ incubator at 38.5°C and 5% CO ₂. Matured COC s were rinsed twice in fertilization TALP and placed in the pre‐warmed fertilization medium without sperm. Cryopreserved buffalo semen was thawed at 37°C for 30 s and processed through Sephadex^?, glass wool filtration and swim‐up (control). Total and motile sperm recovery rates were assessed, resuspended in fertilization TALP and incubated for 15–20 min in CO ₂ incubator. Samples prepared by each method were divided into two aliquots: one aliquot was studied for sperm quality (progressive motility, membrane integrity, viability, liveability), while the other was subjected to co‐incubation with sets of 10–15 in vitro matured oocytes. Data on sperm quality were analysed by ANOVA , while in vitro fertilizing rates were compared by chi‐squared test using SPSS ‐20. Least significant difference (LSD ) test was used to compare treatment means. Glass wool filtration yielded higher total and motile sperm recovery rate, while Sephadex^? filtration improved (p  <  .05) sperm quality (progressive motility, membrane integrity, viability, liveability). Sperm preparation through Sephadex filtration yielded higher in vitro fertilization rate in terms of cleavage rate compared to glass wool filtration and swim‐up (control). In conclusion, cryopreserved Nili‐Ravi buffalo sperm selected through Sephadex filtration showed improved quality and yielded better fertilization rates (cleavage rate) of in vitro matured/fertilized oocytes. Sephadex filtration could be a promising technique for use in in vitro fertilization in buffalo.     

Pharmacokinetics of intravenous gentamicin in healthy young‐adult compared to aged alpacas

The study objective was to evaluate the effects of age on aminoglycoside pharmacokinetics in eight young‐adult (<4 years) and eight aged (≥14 years) healthy alpacas, receiving a single 6.6 mg/kg intravenous gentamicin injection. Heparinized plasma samples were obtained at designated time points following drug administration and frozen at ?80°C until assayed by a validated immunoassay (QMS ^®). Compartmental and noncompartmental analyses of gentamicin plasma concentrations versus time were performed using WinNonlin (v6.4) software. Baseline physical and hematological parameters were not significantly different between young and old animals with the exception of sex. Data were best fitted to a two‐compartment pharmacokinetic model. The peak drug concentration at 30 min after dosing (23.8 ± 2.1 vs. 26.1 ± 2 μg/ml, p = .043 ) and area under the curve (70.4 ± 10.5 vs. 90.4 ± 17.6 μg hr/ml, p = .015 ) were significantly lower in young‐adult compared to aged alpacas. Accordingly, young alpacas had a significantly greater systemic clearance than older animals (95.5 ± 14.4 and 75.6 ± 16.1 ml hr^?1 kg^?1; p = .018 ), respectively). In conclusion, a single 6.6 mg/kg intravenous gentamicin injection achieves target blood concentrations of >10 times the MIC of gentamicin‐susceptible pathogens with MIC levels ≤2 μg/ml, in both young‐adult and geriatric alpacas. However, the observed reduction in gentamicin clearance in aged alpacas may increase their risk for gentamicin‐related adverse drug reactions.     

Influence of different anaesthetic protocols over the sperm quality on the fresh,chilled (4°C) and frozen‐thawed epididymal sperm samples in domestic dogs

This study assessed the influence of three different anaesthetic protocols on semen quality obtained from the epididymis. Sixty male dogs undergoing to routine sterilization were assigned to three anaesthetic protocols: thiopental group (TG, n = 20), propofol group (PG, n = 20) and ketamine–dexmedetomidine group (KDG, n = 20). Immediately after orchidectomy, the cauda epididymides and vas deferent ducts were isolated and then a retrograde flushing was performed to collect spermatozoa. In experiment 1, after the initial evaluation of the semen (sperm concentration, sperm motility and the percentages of live spermatozoa, abnormal spermatozoa and acrosome membrane integrity), semen samples were diluted in Tris‐glucose‐egg yolk extender and chilled for 48 hr, and the sperm motility was assessed at 6, 24 and 48 hr. In experiment 2, semen samples were diluted in Tris‐glucose‐egg yolk extender and chilled for 24 hr, and then samples were frozen in two extenders with different glycerol concentrations, to reach a final concentration of 50–100 × 10⁶ spermatozoa ml^?1, 20% egg yolk, 0.5% Equex and 4% and 5% glycerol, respectively. Mean values of total sperm concentration, sperm viability and the percentages of intact acrosome and abnormal spermatozoa were not significantly different between experimental groups, and therefore, the anaesthetic protocols assessed did not affect sperm parameters mentioned above. However, our study confirmed a detrimental effect of the use of thiopental (TG) over the total sperm motility (p < 0.05) and progressive sperm motility (p < 0.05) of the fresh and chilled epididymal sperm samples. The anaesthetic protocols including the application of propofol or ketamine–dexmedetomidine can be used to recover sperm in domestic canids without significant changes in sperm quality compared when semen is collected routinely and these techniques could be applicable to endangered wild canids.     

Effect of Antioxidants During Bovine In Vitro Fertilization Procedures on Spermatozoa and Embryo Development

Increased amounts of reactive oxygen species (ROS) during in vitro fertilization (IVF) may cause cytotoxic damage to gametes, whereas small amounts of ROS favour sperm capacitation. The aim of this study was to investigate the effect of antioxidants [50 μ m β-mercaptoethanol (β-ME) and 50 μ m cysteamine (Cyst)] or a pro-oxidant (5 m m buthionine sulfoximine) on the quality and penetrability of spermatozoa into bovine oocytes and on the subsequent embryo development and quality when added during IVF. Sperm quality, evaluated by the integrity of plasma and acrosomal membranes, and mitochondrial function, was diminished (p < 0.05) after 4-h culture in the presence of antioxidants. Oocyte penetration rates were similar between treatments (p > 0.05), but antioxidants adversely affected the normal pronuclear formation rates (p < 0.05). The incidence of polyspermy was high for β-ME (p < 0.05). No differences were observed in cleavage rates between treatments (p > 0.05). However, the developmental rate to the blastocyst stage was adversely affected by Cyst treatment (p < 0.05). The quality of embryos that reached the blastocyst stage, evaluated by total, inner cell mass (ICM) and trophectoderm cell numbers and ICM/total cell ratio was unaffected (p > 0.05) by treatments. The results indicate that ROS play a role in the fertilizing capacity in bovine spermatozoa, as well as in the interaction between the spermatozoa and the oocytes. It can be concluded that supplementation with antioxidants during IVF procedures impairs sperm quality, normal pronuclear formation and embryo development to the blastocyst stage.