Comparison of two methods of seminal plasma removal on buffalo (Bubalus bubalis) sperm cryopreservation

Cryopreservation causes damage to spermatozoa, and methods minimizing this damage are therefore needed. Although much discussed, seminal plasma removal has become an alternative to improve sperm quality and viability after freezing and has been applied to different species in attempt to obtain good results. The objective of this study was to evaluate semen quality in buffaloes submitted to two methods for seminal plasma removal (filtration and centrifugation). Semen samples were collected from seven Murrah buffalo bulls (Bubalus bubalis) once a week for 8 weeks. Each ejaculate was divided into three groups: control (presence of seminal plasma), centrifugation and filtration. Sperm kinetics was evaluated with the computer‐assisted sperm analysis (CASA) system. Plasmalemma and acrosomal membrane integrity, mitochondrial membrane potential and reactive oxygen species (ROS) were measured by flow cytometry, and lipid peroxidation was evaluated by the thiobarbituric acid reactive substances (TBARS) assay. Seminal plasma removal did not improve sperm kinetics compared to the control group. Centrifugation increased the number of cells with damaged acrosomal membranes (0.77 ± 0.05) and filtration caused greater plasmalemma and acrosomal membrane damage (22.18 ± 1.07). No difference in the mitochondrial membrane potential was observed between groups. In contrast, ROS production was higher in the centrifugation group compared to the control and filtration groups, although no differences in TBARS formation were detected. In conclusion, seminal plasma removal did not improve the quality of thawed buffalo semen compared to control in terms of sperm kinetics, membrane integrity, mitochondrial membrane potential or lipid peroxidation.     

Estimation of endogenous levels of osteopontin,total antioxidant capacity and malondialdehyde in seminal plasma: Application for fertility assessment in buffalo (Bubalus bubalis) bulls

This study was attempted to identify subfertile bulls by quantifying the endogenous levels of osteopontin (OPN), total antioxidant capacity (TAC) and malondialdehyde (MDA) in seminal plasma of buffalo bulls. On the basis of conception rate, buffalo bulls were classified into two groups: high‐fertile (conception rate >50%) and subfertile bulls (conception rate <40%). A total of 100 ejaculates (10 ejaculates from each bull) were collected through artificial vagina method. The concentration of OPN, TAC and catalase (CAT) of high‐fertile bulls was found to be higher (p < .05) than that of subfertile bulls. Further, MDA level in seminal plasma was found to be lower (p < .05) in high‐fertile bulls compared with subfertile bulls. The fertility status had no effect on the superoxide dismutase (SOD) concentration in seminal plasma of both the groups. The levels of OPN (r = .678, p = 0.013) and TAC (r = .648, p = .042) were found to be positively correlated with bull fertility and the level of MDA (r = ?.718, p = .019) was found to be negatively correlated with bull fertility. However, the fertility of bulls was not found to be significantly correlated with SOD, CAT and sperm motility. In conclusion, seminal OPN, TAC and MDA tended to be more realistic in identification of subfertile bulls from breeding herds.     

Effect of Seminal Plasma on Post‐Thaw Quality and Functionality of Corriedale Ram Sperm Obtained by Electroejaculation and Artificial Vagina

We have already shown that seminal collection method affects seminal plasma composition and sperm quality in Corriedale rams. In this study, we evaluated the effect of seminal plasma collected by electroejaculation or artificial vagina on sperm resistance to cryodamage. Seminal plasma of five rams of the Corriedale breed collected by artificial vagina or electroejaculation was added before freezing to sperm cells collected by the two methods, and post‐thaw quality parameters were evaluated. We found that seminal plasma has no effect on sperm resistance to cryodamage. However, we observed significantly higher percentages of sperm with intact and functional plasma membrane, intact acrosome and greater fertilizing potential after thawing in samples obtained by electroejaculation. This study demonstrates that sperm collected by electroejaculation are more resistant to damage caused by cryopreservation than those collected by artificial vagina.     

The addition of calcium ion chelator,EGTA to thawing solution improves fertilizing ability in frozen–thawed boar sperm

In our previous study, seminal plasma effectively suppressed the induction of sperm to capacitation‐like status and acrosome loss during the thawing process. However, because boar seminal plasma is contaminated with various kinds of bacteria and/or viruses, it is necessary to develop a thawing solution without animal‐derived materials. In this study, we focused on the increase of intracellular Ca²⁺ ([Ca²⁺]_i) in sperm after thawing and the negative effects of sperm qualities. After thawing, the fluorescent intensity of [Ca²⁺]_i indicator, Fluo‐3/AM, and the level of phosphorylated tyrosine residue of protein were increased in the sperm. Next, we investigated whether the addition of Ca²⁺ chelators (ethylenediaminetetraacetic acid (EDTA) and ethyleneglycoltetraacetic acid (EGTA)) improved post‐thawed sperm motility. When the frozen–thawed sperm were treated with 6 mmol/L EDTA + 6 mmol/L EGTA, sperm motility was significantly increased as compared with control (6 mmol/L EDTA alone) at all incubation periods (P < 0.05). The combinational treatment significantly suppressed the elevation of [Ca²⁺]_i and the tyrosine phosphorylation, which improved the acrosomal status and fertilizing ability in vitro. Furthermore, when the thawing method was applied for artificial insemination, the fertilization rate was significantly higher than control (P < 0.05, 33% vs. 82%). Thus, we conclude that the addition of EDTA + EGTA to thawing solution is a beneficial tool for artificial insemination using frozen–thawed boar sperm.     

Preservation of Epididymal Stallion Sperm in Liquid and Frozen States: Effects of Seminal Plasma on Sperm Function and Fertility

Three separate experiments were conducted to improve preservation of stallion epididymal sperm. In the first one, two different cooling extenders (Kenney and Gent) were compared. Sperm viability and motility patterns were assessed in 10 different epididymal sperm samples after 0 hours, 24 hours, 48 hours, 72 hours, and 96 hours of preservation at 4°C. No significant differences were observed in any of the evaluated parameters either between extenders or throughout the storage period. The second set of experiments was designed to determine whether supplementing thawing medium (INRA Freeze) with seminal plasma had any impact on the quality of frozen-thawed epididymal sperm. Ten epididymal frozen-thawed sperm samples coming from separate stallions were used and different functional parameters (sperm membrane integrity and lipid disorder, motility, intracellular Ca²⁺ levels, and intracellular concentrations of peroxides and superoxides) were evaluated after incubation with or without 50% seminal plasma. Supplementing thawing medium with seminal plasma had no impact on sperm function and survival. The third experiment was an in vivo study. Twenty-five mares were inseminated with epididymal frozen-thawed sperm and seminal plasma, and 21 were bred with epididymal frozen-thawed sperm only. Pregnancy rates obtained for mares artificially inseminated with epididymal frozen-thawed sperm and seminal plasma were significantly (P < .05) higher than those observed when seminal plasma was not infused (64% vs. 19%). Taken together, our data indicate that the quality of epididymal stallion sperm can be maintained at 4°C for up to 96 hours. In addition, not only does supplementing frozen-thawed epididymal sperm with seminal plasma have any damaging effect on their quality but it may also improve pregnancy rates after artificial insemination.     

Lipid and protein oxidation levels in spermatozoa and seminal plasma of Asian Elephants (Elephas maximus) and their relationship with semen parameters

Peroxidation damage to spermatozoa and seminal plasma has an important role in sperm quality. Thus, the objective of this study was to determine the levels of lipid and protein oxidation in spermatozoa and seminal plasma of Asian elephants (Elephas maximus) with varying percentage of progressive motility. Lipid and protein oxidation was measured by the thiobarbituric acid‐reactive species (TBARS) assay and the 2, 4‐dinitrophenylhydrazine (DNPH) carbonyl groups assay, respectively. Fresh semen samples were collected from Asian elephants and classified according to the percentage of motile spermatozoa into good (>60%) and poor (≤20%) motility. Results revealed that seminal plasma malondialdehyde (MDA) and seminal plasma protein carbonyls (PCs) were significantly higher in poor motility than in good motility (p < .05). The MDA and PC levels in seminal plasma were negatively correlated with the percentages of progressive motility (p < .05). In addition, the negative correlation between sperm concentration and seminal plasma MDA level was investigated (p < .05). The sperm viability was also negatively correlated with sperm PC level (p < .05). This study indicated that lipid and protein oxidation has deleterious effect on semen quality of Asian elephants.     

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.     

In Vitro Evaluation of Liquid‐stored Buffalo Semen at 5°C Diluted in Soya Lecithin Based Extender (Bioxcell®), Tris‐Citric Egg Yolk,Skim Milk and Egg Yolk‐Citrate Extenders

This study was designed to compare the quality of liquid‐stored buffalo bull spermatozoa in soya lecithin based extender Bioxcell^® (BIOX), milk (MILK), tris‐citric egg yolk (TEY) and egg yolk‐citrate (EYC) extender at 5°C. Semen was collected from five Nili‐Ravi buffalo (Bubalus bubalis) bulls of 6–7 years of age with artificial vagina over a period of 3 weeks (two consecutive ejaculates once in a week). Semen ejaculates having more than 60% motility were pooled, split into four aliquots, diluted (37°C; 10 × 10⁶ motile spermatozoa/ml), cooled from 37 to 5°C in 2 h (0.275°C/min) and stored for 5 days. Sperm motility, viability, plasma membrane integrity (PMI) and normal acrosomal ridge were studied at first, third and fifth day of storage. Higher values of progressive sperm motility (%), sperm viability (%), sperm PMI (%) and normal apical ridge (%) were observed in BIOX, MILK and TEY extenders at first, third and fifth day of storage than EYC extender. Progressive sperm motility, sperm viability and sperm PMI in BIOX^® extender were not different from MILK and TEY extenders at 1st and third day storage period. However, at fifth day of storage, the values for these parameters remained significantly higher (p < 0.05) in BIOX^® compared with MILK, TEY and EYC extenders. At fifth day of storage, the semen quality parameters for Bioxcell^® were comparable to those with MILK and TEY extenders at third day of storage. In conclusion, motility, viability and PMI of buffalo bull spermatozoa remained similar in Bioxcell^®, milk and TEY extender at first and third days of storage at 5°C. Yet, the values for the aforementioned parameters in Bioxcell^® were higher compared with milk, TEY and EYC extender at fifth day of storage at 5°C.     

Influence of seminal plasma during different stages of bovine sperm cryopreservation

This study aimed to evaluate the effect of seminal plasma on bovine sperm cryopreservation and to assess the integrity of plasma and acrosomal membranes, mitochondrial potential, remodelling of F-actin cytoskeleton and sperm chromatin fragmentation during the cooling, equilibrium and freezing/thawing stages. Six ejaculates collected from seven Nelore bulls (n = 42) were used in this study. Each ejaculate was divided into two aliquots (with seminal plasma = SP group; without seminal plasma = NSP group) and packed to a final concentration of 50 × 10⁶ sperm per straw. Statistical analyses were performed using SAS software (version 9.3), and p ≤ .05 was considered significant. A time effect was observed for all sperm characteristics (p < .05), except for chromatin fragmentation (p > .05). The presence of seminal plasma better preserved the acrosomal integrity (SP = 75.2% and NSP = 71.7%; p < .05) and also provided lower F-actin remodelling during cryopreservation process (SP = 29.9% and NSP = 32.4%; p < .05). Regarding to the cryopreservation stages, it was observed that cooling step induced higher remodelling of F-actin than the equilibrium and freezing/thawing stages (56.3%, 32.2% and 23.9%, respectively; p < .05). The equilibrium step had minor influence on overall sperm characteristics while the freezing/thawing stage was responsible for the highest percentage of damage in plasma membrane (−65.2%), acrosomal membrane (−34.0%) and mitochondrial potential (−48.1%). On the other hand, none of the cryopreservation stages affected chromatin integrity. It was concluded that the presence of seminal plasma provides increased acrosomal integrity and reduced remodelling of F-actin cytoskeleton. Higher F-actin remodelling is observed after the cooling step while the freezing/thawing step is most damaging to sperm membranes and mitochondrial potential during bovine sperm cryopreservation.     

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.     

Development of an in vitro oviduct epithelial explants model for studying sperm–oviduct binding in the buffalo

In this study, we developed an in vitro model for studying sperm–oviduct binding in the buffalo. Oviduct explants were prepared by overnight culture of epithelial cells in TCM‐199 medium under 5% CO₂ at 38.5 °C. Cryopreserved spermatozoa from buffalo bulls (n = 4) were incubated with the oviduct explants, and the sperm–oviduct explants complex was stained with JC‐1. The effect of sperm concentration (2, 3 and 4 million), size of the oviduct explants (<0.2, 0.2–0.3, 0.3–0.4 and >0.4 mm²) and time of incubation (1 hr and 4 hr) on binding index (BI—number of sperm bound to unit area of explants) was studied. No significant difference was observed in the BI among <0.2, 0.2–0.3 and 0.3–0.4 mm² size of explants; however, the BI decreased significantly (p < .05) when the size of explants exceeded 0.4 mm². The BI decreased significantly (p < .05) when the sperm concentration was increased to 4 million, while the duration of incubation did not have any significant effect on the BI. The interaction of bulls with explants size, sperm concentration and incubation time was not significant. The developed assay has the potential to be used as an in vitro model for studying sperm–oviduct binding in the buffalo.     

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.     

Total antioxidant capacity and protein peroxidation intensity in seminal plasma of infertile and fertile dogs

The aim of this study was to evaluate the total antioxidant capacity and protein peroxidation intensity in seminal plasma of infertile and fertile dogs. The study was conducted on 10 infertile and 10 fertile dogs of various breeds. Infertility was defined as conception failure at least three matings with different bitches. Semen was collected by manual manipulation. The sperm concentration and motility parameters were evaluated using CASA Hamilton Thorne, Vers. IVOS 12.3. The morphology of spermatozoa and the percentage of live and dead sperm cells were assessed microscopically, total antioxidant capacity and the content of SH‐groups in seminal plasma were determined spectrophotometrically, the contents of protein peroxidation markers in seminal plasma, bityrosine and formylokinurenine, were determined using spectrofluorimetric methods. Sperm concentration and total sperm count were significantly (p < 0.05) lower in infertile dogs than in fertile dogs (99.92 ± 3 0.05 × 10⁶/ml vs. 282.07 ± 48.27 × 10⁶/ml; 214.19 ± 114.74 × 10⁶ vs. 747.57 ± 210.94 × 10⁶, respectively). The percentage of spermatozoa with normal morphology and the most determined motility parameters differed significantly (p < 0.05) between both groups. The mean values of total antioxidant capacity in the seminal plasma were significantly (p < 0.05) lower (19.95 ± 20.94 vs. 25.66 ± 23.18 µmol/g protein), whereas the mean contents of bityrosine and formylokinurenine in seminal plasma were significantly (p < 0.05) higher in infertile dogs than in fertile dogs (3.71 ± 4.83 µg/mg protein vs. 1.55 ± 2.00 µg/mg protein and 0.37 ± 0.45 µg/mg protein vs. 0.14 ± 0.08 µg/mg protein, respectively). In conclusion, the obtained results suggest that the poor semen quality and infertility in dogs could be associated with lowered total antioxidant capacity and increased protein peroxidation in seminal plasma as a consequence of oxidative stress.     

Serum kisspeptin: New possible biomarker for sexual behaviour and sperm concentration in buffalo bulls

The study was designed to decipher the inter-relationship between peripheral hormones (kisspeptin and testosterone), sexual behaviour and seminal variables of Murrah buffalo bulls (n = 134). In this study, we recorded that 13%, 37%, 40%, 6% and 4% Murrah buffalo bulls had reaction time of <30, 31–60, 61–180, 181–300 and >300 s, respectively. Further, it was observed that 4%, 85% and 10% buffalo bulls were sexually aggressive, active and dull, respectively, during semen collection. The courtship behaviour was not found to be desirable for the bulls used for the semen collection. Mean of ejaculate volume, sperm concentration and mass motility (0–5 scale) were 3.57 ml, 977.11 million/ml, 2.7, respectively. Correlation studies revealed that the reaction time was positively correlated with courtship behaviour and body weight, and negatively correlated with sexual aggressiveness and sperm concentration. Serum kisspeptin in buffalo bulls, measured for the first time, was found to 3.8 ± 0.7 ng/ml. Serum kisspeptin and testosterone level are negatively correlated to each other and kisspeptin level influenced the sexual behaviour (reaction time, sexual aggressiveness and penile erection) of study bulls. Serum kisspeptin was higher in the buffalo bulls with higher sperm concentration indicating its role in spermatogenesis. In conclusion, for the first time basic information related to sexual behaviour of Murrah buffalo bulls in large population along with its inter-relationship with peripheral hormones (kisspeptin and testosterone) has been documented.     

Electroejaculation Increases Low Molecular Weight Proteins in Seminal Plasma Modifying Sperm Quality in Corriedale Rams

This study was conducted to evaluate the effect of seminal collection method (artificial vagina or electroejaculation) on the protein composition of seminal plasma and sperm quality parameters in Corriedale rams. To address this question, we assessed the effect of seminal collection method on motility, plasma membrane integrity and functionality, mitochondrial functionality and the decondensation state of nuclear chromatin in sperm cells. Volume, pH, osmolarity, protein concentration, total protein content and protein profile using sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS‐PAGE) and 2‐D polyacrylamide electrophoresis of seminal plasma collected with artificial vagina and electroejaculation were also analysed. The main findings from this study were that ejaculates obtained with electroejaculation had (i) a higher number of spermatozoa with intact plasma membrane and functional mitochondria and (ii) a higher proportion of seminal plasma, total protein content and relative abundance of low molecular weight proteins than ejaculates obtained with artificial vagina. Five of these proteins were identified by mass spectrometry: binder of sperm 5 precursor; RSVP14; RSVP22; epididymal secretory protein E1 and clusterin. One protein spot with molecular weight of approximately 31 kDa and isoelectric point of 4.8 was only found in the seminal plasma from electroejaculation.     

Influence of Inseminate Components on Porcine Leucocyte Migration In Vitro and In Vivo after Pre- and Post-Ovulatory Insemination

A post‐breeding migration of leucocytes (PMN) into the uterus is considered to be an important reason for sperm losses. Minimizing such effects may be necessary for successful insemination with low sperm numbers, as required with sex‐sorted spermatozoa. We examined the magnitude of PMN influx 3 h after pre‐ or post‐ovulatory insemination with various combinations of seminal plasma (SP), semen extender Androhep? (AH; Minitüb, Tiefenbach, Germany) and sperm preparations (S). Pre‐ovulatory inseminations with preparations containing 98% AH caused a massive influx of PMN, independent of whether spermatozoa were present (628 ± 189 × 10⁶ leucocytes/uterine horn) or not (580 ± 153 × 10⁶). Post‐ovulatory, 98% AH caused a comparable immigration only in the absence of sperm cells (AH: 569 ± 198 × 10⁶, AH+S: 162 ± 102 × 10⁶). The presence of SP significantly dampened the numbers of recruited uterine leucocytes. The reaction to all inseminates containing 98% SP both with and without spermatozoa, used before ovulation (SP: 14 ± 6 × 10⁶, SP+S: 73 ± 27 × 10⁶) and after ovulation (SP: 60 ± 32 × 10⁶, SP+S: 51 ± 33 × 10⁶) did not differ significantly from controls using phosphate buffered saline (PBS) (pre‐ovulatory: 1 ± 1 × 10⁶, post‐ovulatory: 11 ± 9 × 10⁶). Quantitative in vitro transmigration assays with blood‐derived PMN proved that AH‐induced leucocyte migration into the uterus to be not as a result of direct chemotaxis, because, on account of the chelator citrate, AH significantly inhibited the transmigration towards recombinant human Interleukin‐8 (rhCXCL8) (AH: 14 ± 5% migration rate vs controls: 37 ± 6%, p < 0.05). Supernatants of spermatozoa incubated in PBS for 1, 12 or 24 h showed neither chemoattractive nor chemotaxis‐inhibiting properties. SP at ≥0.1% [v/v] significantly inhibited the in vitro transmigration of PMN. With respect to in vivo migration of neutrophils, the striking difference in the results between semen extender and seminal plasma suggests that adaptation of extender composition is needed to reflect more closely the in vivo regulatory potential of natural seminal plasma.     

Selection of red deer spermatozoa with different cryoresistance using density gradients

The objective of sperm selection media is selecting the best spermatozoa and to remove seminal plasma and diluent for using them in assisted reproductive techniques. It is known that individuals show different cryoresistance in response to the same freezing procedure. Our hypothesis was that the efficacy of selection media could be dissimilar for samples with different sperm quality after thawing. Epididymal sperm samples from mature Iberian red deer were collected and frozen. Males were classified as with high post‐thaw sperm quality when sperm motility (SM) ≥ 70%, or as with low post‐thaw sperm quality when SM ≤ 69%. Samples were centrifuged using the following density gradients (DG): Percoll^®, Puresperm^® and Bovipure^?, and several functional sperm parameters were assessed after sperm selecting and washing. Males classified with high sperm quality had higher post‐thawing values (p > .05) for all parameters evaluated, except for linearity index, than those categorized as low sperm quality. After selection, some sperm characteristics improved (viability, apoptosis and mitochondrial activity) for both groups, showing the males with high sperm quality higher values in all sperm parameters except for kinematic traits and DNA fragmentation index (%DFI), regardless of DG. Bovipure^? yield lower values of sperm motility, viability, apoptosis and mitochondrial activity in relation to Percoll^® and Puresperm^® considering both quality groups. There was an interaction between the type of DG and sperm quality group for sperm viability (p = .040) and apoptosis (p = .003). Thus, Percoll^® selected less live and more apoptotic spermatozoa than Puresperm^® and Bovipure^? for males with low sperm quality. In conclusion, the DG are more efficient selecting spermatozoa from samples with high sperm quality, acting differently depending on initial sperm quality.     

Cryopreservation of Nili‐Ravi buffalo (Bubalus bubalis) semen in AndroMed® extender; in vitro and in vivo evaluation

The study was designed to evaluate AndroMed^® for the freezability and fertility of Nili‐Ravi buffalo semen. Semen was collected from four adult Nili‐Ravi buffalo (Bubalus bubalis) bulls for 3 weeks (replicate). Semen ejaculates from each buffalo bull were divided into three aliquots. One aliquot was used for evaluation of motility, plasma membrane integrity, livability, viability, DNA integrity and normal apical ridge. Remaining two aliquots were diluted (37°C; 50 × 10⁶ spermatozoa/ml) in tris‐citric egg yolk or AndroMed^® extender and cryopreserved in 0.5 ml French straws. After thawing, per cent post‐thaw motility (47.9 ± 0.8, 49.2 ± 1.7), plasma membrane integrity (44.4 ± 1.2, 46.8 ± 1.8) and normal apical ridge (81.4 ± 0.3, 83.2 ± 0.3) were recorded similar (p > .05) in tris‐citric egg yolk and AndroMed^® extender. Higher (p < .05) percentage of sperm livability (70.5 ± 1.4 and 64.4 ± 1.0), viability (67.5 ± 1.5 and 61.5 ± 0.6) and DNA integrity (97.0 ± 0.3 and 93.4 ± 0.21) were recorded in AndroMed^® compared to tris‐citric egg yolk post‐thaw. Values for all the aforementioned spermatozoal quality parameters were observed lower (p < .05) in frozen‐thawed compared to fresh semen irrespective of the experimental extenders. Fertility rates of buffalo semen did not differ (p > .05) either cryopreserved in tris‐citric egg yolk or AndroMed^® extender (45.5% vs. 49%). It is concluded that AndroMed^® is capable in protecting the buffalo bull sperm during freeze‐thawing process and can be adopted safely for routine use replacing the tris‐citric egg yolk extender in artificial insemination programme.     

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.     

Exploring the metabolome of seminal plasma in two different horse types: Light versus draft stallions

The application of the ‘omics’ studies in the field of animal reproduction has been aimed at identifying novel biomarkers of fertility since the last few years. When assessing reproductive efficiency in horses, breed should also be taken into account as it can influence semen quality and fertility. Considering the growing interest in metabolomic analysis to evaluate male fertility, we aimed to investigate the metabolomic profile of seminal plasma in two different horse breeds. Twelve healthy stallions, n.6 American Quarter Horse (AQH) and n.6 Italian Draft Horse (IDH) stallions, regularly used for artificial insemination, were included in the study. Two semen collections, performed 30-day apart, were considered for the assessment of semen parameters including gel-free volume, spermatozoa (spz) concentration, spz progressive motility and seminal plasma analysis by ¹H-NMR.Semen characteristics differed between IDH and AQH (p < .05) as well as the first cycle conception rate that was higher in AQH than IDH (p = .001). Metabolomic analysis quantified 56 molecules in equine seminal plasma, with 11 metabolites showing different concentrations in IDH compared to AQH (p < .05).This study provided evidence of differences in seminal plasma metabolites' concentrations between studied horse types, highlighting specific metabolomic fingerprints characterizing AQH and IDH sperm.