In Vitro Maturation and Fertilization of Buffalo Oocytes: Effects of Storage of Ovaries, IVM Temperatures, Storage of Processed Sperm and Fertilization Media

Studies were conducted to examine the possibility of preserving slaughterhouse‐derived buffalo ovaries at 4°C for 0 (control), 12 and 24 h to maintain the developmental competence of the oocytes (experiment 1), to assess the effect of incubation temperature during oocyte maturation on rates of in vitro maturation (IVM) and in vitro fertilization (IVF) of buffalo oocytes and embryo development (experiment 2), and to examine the effect of storage at 25°C for 0 (control), 4 and 8 h of frozen–thawed buffalo sperm and BO and H‐TALP as sperm processing and fertilization media on cleavage and embryo development in vitro of buffalo oocytes (experiment 3) in order to optimize the IVF technology in buffalo. Results suggested that storage of ovaries at 4°C for 12 or 24 h significantly (p < 0.05) reduced the developmental potential of oocytes. Incubation temperatures during the IVM influenced the fertilization rate but had no significant effect on maturation and subsequent embryo development. The incubation temperature of 38.5°C during IVM was found to be optimum for embryo production in vitro. Storage of frozen–thawed sperm at 25°C for 8 h significantly (p < 0.05) decreased its ability to cleave the oocytes. Sperm processed in BO medium had significantly (p < 0.05) higher ability to cleave the oocytes than the H‐TALP medium.     

Application of Techniques for Sperm Selection in Fresh and Frozen-Thawed Stallion Semen

The objective of this research was to improve the techniques in processing chilled and frozen‐thawed horse semen. In a preliminary experiment (Exp. I), different techniques for sperm selection and preparation [Swim‐up, Glass wool (GW) filtration, Glass wool Sephadex (GWS) filtration; Percoll] were tested for their suitability for equine spermatozoa and results were compared with the routine procedure by dilution (Exp. I). In the main experiment (Exp. II), two sperm preparation techniques (GWS, Leucosorb^®) refering to the results of Exp. I and a previous study of our group (Pferdcheilkunde 1996 12, 773) were selected for processing complete ejaculates either for cooled‐storage or cryopreservation. In a third experiment (Exp. III), pregnancy rates from inseminations with semen processed according to the techniques tested in Exp. II were compared with those obtained with semen processed according to routine procedures. In Exp. I (six stallions, six ejaculates/stallion), between 48 and 92% of spermatozoa were lost following the different sperm selection procedures (p < 0.05). Preparation of sperm increased percentage of progressively motile spermatozoa (pms) [Swim‐up, GW, GWS vs dilution, Percoll (p < 0.05)] and decreased percentage of sperm head abnormalities [Swim‐up, GW, GWS vs dilution, Percoll (p < 0.05)] probably by not improving the quality of individual cells, but by elimination of spermatozoa of inferior quality. In Exp. II (eight stallions, three ejaculates/stallion) Leucosorb^® and GWS procedures allowed the filtration of large volumes (extended ejaculates) for routine laboratory practice. GWS and Leucosorb^® filtration resulted in increased motility, membrane integrity and sperm viability after storage of spermatozoa until 48 h at +5°C when compared with control (diluted) and centrifuged semen (p < 0.05). Significantly more spermatozoa were recovered after centrifugation (87.8 ± 15.4%) compared with GWS (63.5 ± 18.6%) and Leucosorb^® filtration (53.6 ± 22.3%). GWS or Leucosorb^® procedure resulted in successful cryopreservation of stallion semen without centrifugation for removal of seminal plasma. The per cycle conception rate of inseminated mares using 200 × 10⁶ pms transferred within 8 h after collection of semen was not affected by GWS filtration or Leucosorb^® separation when compared with centrifugation (n.s.; Exp. III). In conclusion, GWS and Leucosorb^® filtration results in the improvement of semen quality and should be considered as a method for stallion semen processing. Additional studies are needed for the evaluation of potentially higher fertilizing ability of stallion spermatozoa separated by techniques for sperm selection.     

Effects of Matrix Filtration of Low-Quality Boar Semen Doses on Sperm Quality

The aim of this work was to develop a method to enhance the sperm parameters of ejaculates with low sperm quality from Piétrain boars. Seminal doses were filtered through columns of DEAE Sephadex (length 2.5 ± 0.5 cm), CM Sephadex (length 5 ± 0.5 cm), glass wool (length 2 ± 0.5 cm) or glass bead (length 10 ± 0.5 cm), with an exit flow rate of 1 ml/40 s in all cases. For each male, 10 ml of the sperm cell-rich fraction diluted at 1 : 6 were filtered. Sperm quality was assessed before and after filtration. Sperm morphology, sperm motility and sperm concentration were determined using the computer program sca ^® 2002 Production, and sperm viability was evaluated by fluorescence multistaining. Osmotic resistance test and hyperosmotic resistance test were used to determine the osmotic resistance of spermatozoa, whereas l -lactate production estimated the metabolic activity. Results showed a decrease of sperm concentration and osmotic resistance of spermatozoa after filtration in the four matrixes. However, an increase in the frequency of viable spermatozoa with intact acrosome after filtration in glass bead columns and an increase of morphologically normal spermatozoa after filtration in Sephadex CM-50, glass wool and glass bead columns were observed. Despite the decrease in the frequency of progressive motile spermatozoa, l -lactate production and mitochondrial sheath integrity maintained constant after filtration. Our findings indicate that column filtration is an effective method to enhance the sperm quality by selecting viable and morphologically normal spermatozoa without altering DNA, plasma membrane, mitochondrial sheath integrity or inducing premature acrosome reaction.     

A comparative study of Sephadex, glass wool and Percoll separation techniques on sperm quality and IVF results for cryopreserved bovine semen

The aim of this study was to compare the effects of spermatozoa separation techniques on sperm quality and in-vitro fertilization (IVF) results for cryopreserved bovine semen. Sephadex, glass wool and Percoll gradient separation techniques were used for sperm separation and sperm motility, morphology and membrane integrity were evaluated before and after separation. Also, cleavage and blastocyst developmental rate were investigated after IVF with sperm recovered by each separation technique. The motility of samples obtained by the three separation techniques were greater compared to the control samples (p < 0.05). The percentage of spermatozoa with intact plasma-membrane integrity, identified by 6-carboxyfluoresceindiacetate/propidium iodide fluorescent staining and the hypo-osmotic swelling test, was highest in the glass wool filtration samples (p < 0.05). The cleavage and blastocyst rate of total oocytes produced from glass wool filtration samples were also higher than the control and Sephadex filtration samples (p < 0.05), but were not significantly different from Percoll separation samples. However, a significantly greater number of cleaved embryos produced by glass wool filtration developed to blastocyst stage than those produced by Percoll separation (p < 0.05). These results indicate that spermatozoa with good quality can be achieved by these three separation techniques and can be used for bovine IVF. In particular, it suggests that glass wool filtration would be the most effective method of the three for improving sperm quality and embryo production for cryopreserved bovine spermatozoa.     

Short‐Term Storage and Swim‐Up Selection Do Not Affect the X/Y Ratio in Equine Spermatozoa

The standard procedure of artificial insemination with fresh equine spermatozoa involves short‐term storage (to 48 h at 5°C). This procedure is accompanied by a gradual loss of sperm viability. The aim of this study was to investigate whether the X/Y ratio of equine spermatozoa is affected by short‐term storage and the swim‐up procedure. We used a standard protocol, for short‐term storage (0, 24 and 48 h at 5°C) of stallion semen diluted in the commercial extender EquiPro? (Minitüb GmbH, Tiefenbach, Germany). After each set‐up storage period, the motile fraction of sperm cells was selected by the swim‐up method. The X/Y ratio was evaluated by fluorescence in situ hybridization (FISH) in the fresh, non‐selected sperm, and in motile spermatozoa selected after each of the storage periods. Molecular probes for the equine chromosomes X and Y were used. The X/Y ratio in all sperm samples analysed in this study (fresh and stored) was not different from the theoretical 1 : 1 value. The incidence of chromosomally abnormal sperm cells in the fresh (0.28%) and motile (0.13%) sperm samples was not significantly different. The two approaches (sperm storage up to 48 h and the swim‐up procedure) applied to this study did not affect the X/Y ratio in the motile fraction of equine spermatozoa. This finding does not conform to phenomena described for human and cattle. For this reason, the finding may imply species‐related differences.     

Failure to Remove Bovine Diarrhea Virus (BVDV) from Bull Semen by Swim up and other Separatory Sperm Techniques Associated with in vitro Fertilization

Semen samples, collected from bulls pesistently infected with bovine viral diarrhea virus (BVDV) and containing BVDV (titer 10⁵ - 10⁶TCID₅₀/ml), were subjected to sperm separation procedures (washing, swim up, Percoll gradient, glass wool filtration, glass beads filtration) that are commonly used prior to in vitro fertilization (IVF) to determine if these procedures would yield spermatozoa free from BVDV. The final sperm pellets from frozen and fresh ejaculates were tested for the presence of BVDV by the immunoperoxidase technique; all tests were positive for BVDV in the range of 10³ - 10⁴TCID₅₀/ml). The study shows that when semen containing BVDV at the level of 10⁵ - 10⁶TCID₅₀/ml) is used for IVF, the virus is not completely removed by any of the simple physical methods commonly used to prepare sperm for IVF. Inhalt: Feldversuch das bovine Diarrhoe Virus (BVDV) aus Bullensperma durch Swim up oder andere Trennverfahren in Verbindung mit der In vitro Fertilisation zu entfernen Spermaproben, die von Bullen gewonnen wurden, welche dauerhaft mit dem Virus der bovinen Virus-Diarrhoe (BVDV) infiziert waren und einen Titer zwischen 10⁵ - 10⁶TCID₅₀/ml enthielten, wurden verschiedenen Trennverfahren unterzogen (Waschen, swim up, Percoll gradient, Glaswollenfiltration, Glaskugelfiltration). Diese Verfahren werden üblicherweise für die Vorbereitung zur In vitro Befruchtung verwendet, und es sollte geprüft werden, ob diese Verfahren auch das Sperma von dem BVD-Virus befreien können. Das endgültige Spermapellet von gefrorenen/aufgetauten und frischen Ejakulaten wurde in Gegenwart des BVD-Virus und mit Hilfe der immunoperoxidase Technik getestet. Alle Testergebnisse waren positiv für BVD-Virus in einem Bereich von 10³ - 10⁴ TCID₅₀/ml. Die Studie zeigt, daß Sperma, wenn es 10⁵-10⁶TCID₅₀/ml des Virus enhält, nicht vollständig mit den für die IVF üblichen, einfachen physikalischen Methoden von dem Virus befeit werden kann.     

Effects of Hydrogen Peroxide (H2O2) on Fresh and Cryopreserved Buffalo Sperm Functions During Incubation at 37°C In Vitro

The magnitude of damage to buffalo spermatozoa during incubation with different levels of H₂O₂ was assessed. A total number of 24 ejaculates from four Murrah buffalo bulls were analysed in the study. Each ejaculate was split into two parts (part I and II). Part I was extended in Tris–egg yolk–citrate extender (20% egg yolk:7% glycerol), equilibrated (4 h at 5°C) and cryopreserved in 0.5‐ml French straws and stored in liquid nitrogen. The other part was utilized for fresh semen studies. The sperm in fresh, equilibrated and frozen–thawed semen was separated by centrifugation (1500 g ; 15 min) and were washed with sperm TALP. The sperm cells were re‐suspended in incubation TALP at the rate of 10⁸ sperm cells per millilitre and incubated with 0, 10, 25, and 50 μm H₂O₂ per ml at 37°C. Sperm motility, viability and intact acrosome percentages were assessed at 15‐min intervals up to 60 min of incubation. Lipid peroxidation levels of sperm were assessed at 0 and 60 min of incubation. The results of the experiment revealed that sperm motility decreased drastically during incubation with H₂O₂. Among the different levels of H₂O₂, the 50‐μm H₂O₂‐incorporated group had significantly (p < 0.05) higher malonaldehyde (MDA) level than the other groups. In the 50‐μm H₂O₂‐incorporated group, the MDA levels in fresh, equilibrated and frozen–thawed semen after incubation for 60 min were 961.6 ± 12.7, 991.8 ± 10.3 and 1234.9 ± 9.6 nm per 10⁹ spermatozoa respectively. An inverse relationship was observed between sperm motility, viability, intact acrosome percentages and concentration of H₂O₂ and duration of incubation. The decrease in sperm functions with duration of incubation and concentration of H₂O₂ was significantly (p < 0.05) higher in frozen–thawed than fresh and equilibrated spermatozoa.     

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 relaxin on the motility, acrosome reaction and utilization of glucose of fresh and frozen-thawed bovine spermatozoa

The study was conducted to investigate the effect of relaxin on motility, acrosome reaction (AR), viability and utilization of glucose in fresh and frozen‐thawed bovine spermatozoa. Both semen samples were washed twice through centrifugation (5 min at 600 g), and preincubated for 1 h at 39°C for swim up. The swim‐up separated spermatozoa were resuspended in a sperm Tyrode's albumin lactate pyruvate (Sp‐TALP) medium containing 0 (control) and 40 ng/mL porcine relaxin and incubated for 0–6 h. Sperm motility was determined on the basis of movement quality examined by a phase contrast microscope. Sperm viability and AR were evaluated by using the triple staining technique. The incorporation and oxidation of ¹⁴C‐glucose was assessed by a liquid scintillation counter. Motility was improved (P < 0.05) in both fresh and frozen‐thawed spermatozoa by the addition of relaxin to the Sp‐TALP medium, whereas relaxin showed no significant effect on viability in either fresh or frozen‐thawed spermatozoa. The percentage of AR increased (P < 0.05) when fresh or frozen‐thawed spermatozoa were incubated with relaxin. In contrast, the incorporation and oxidation of ¹⁴C‐glucose increased (P < 0.05) in both kinds of spermatozoa incubated with relaxin. Thus the results demonstrated that the addition of relaxin to the Sp‐TALP medium increased the motility, AR and utilization of glucose in fresh and frozen‐thawed bovine spermatozoa.     

In vitro production of porcine zygotes using intracytoplasmic injection of vitrified sperm

The objectives of this study were to evaluate if vitrified porcine spermatozoa are able to maintain their capacity to produce zygotes in vitro using intracytoplasmic sperm injection (ICSI) and to evaluate the zygote development in two in vitro atmospheric conditions: 5% CO₂ and tri‐gas. A group of porcine oocytes maturated in vitro were injected with vitrified‐warmed sperm (treatment group) and another group, with sperm diluted and conserved at 17°C (control group). To evidence parthenogenetic activation, some oocytes were submitted to a Sham test. The injected oocytes were cultured in G1 medium at 38°C, 100% humidity and 5% CO₂ or tri‐gas. No significant differences (p > .05) were observed in embryo development between the oocytes injected with vitrified‐warmed sperm (31.8%; 36/113), and those injected with semen diluted and conserved at 17°C (35.5%; 32/90), when cultured in 5% CO₂ or under tri‐gas atmosphere (42.9%; 39/91 vs. 34.2%; 26/76, respectively). No significant differences (p > .05) were observed in the percentage of pronuclei (PN) obtained between 5% CO₂ and tri‐gas, within each treatment either. Of the 52 oocytes submitted to the Sham test, only two presented a female PN (activation) indicating that the PN observed in the treatment group were a product of fertilization and not parthenogenetic activation. To conclude, porcine sperm vitrified using spheres, at a concentration of 5 × 10⁶ spermatozoa/ml in TALP medium with 1% bovine serum albumin (BSA), conserve condensed and intact chromatin capable of producing early embryo development up to the pronuclear stage.     

Sperm‐TALP: An Alternative Extender for Retrieving and Diluting Epididymal Sperm in the Domestic Cat

Effects of sperm‐TALP (TALP) on the quality of fresh‐extended and frozen‐thawed epididymal cat sperm were evaluated. The epididymides suspended in Tris–glucose–citrate solution (Tris), a conventional medium, and TALP were cut into small pieces to recover epididymal sperm. In experiment 1, the sperm pellets remained after centrifugation were re‐suspended (1 : 2, v/v) in Tris and TALP. The sperm quality in all four groups, that is, sperm retrieved with Tris (I and II) or TALP (III and IV) and diluted with Tris (I and III) or TALP (II and IV) was assessed. The sperm motility at the 0‐h incubation in TALP–TALP was superior to that of the rest (p < 0.001 to p = 0.04). At the 2‐h incubation, the motility in Tris/TALP–TALP was greater than that in Tris/TALP–Tris (p ≤ 0.001). In experiment 2, after centrifugation, the sperm pellets were added with freezing extenders and frozen. The thawed sperm previously retrieved from the epididymides with Tris and TALP were allotted so as not to further diluted (Tris/TALP–O) and to further diluted (1 : 1, v/v) with Tris (Tris/TALP–Tris) and TALP (Tris/TALP–TALP) and were evaluated the quality. At both incubation times, the motility of frozen‐thawed sperm recovered with TALP (TALP–O/Tris/TALP) was comparable with or significantly higher than that in the Tris groups (Tris–O/Tris/TALP; p = 0.003 to p > 0.05). The motility and viability of thawed sperm in Tris–Tris were significantly decreased during the 2‐h incubation (p = 0.007 for the motility and p = 0.01 for the viability). In both experiments, neither type of diluent (Tris vs TALP) nor incubation time (0 vs 2 h) significantly affected the sperm membrane integrity under hypo‐osmotic condition (p > 0.05). According to beneficial effects on the quality of fresh‐extended and frozen‐thawed sperm demonstrated, sperm‐TALP could be used as an alternative medium for recovering sperm from the epididymides and for diluting epididymal sperm in the domestic cat.     

Effect of Albumin and Polyvinyl Alcohol on the Vitality, Motility and Acrosomal Integrity of Canine Spermatozoa Incubated in vitro

Sperm culture media used for in vitro fertilization (IVF) procedures are important factors concerning the viability, motility and acrosomal integrity of spermatozoa. The aim of this study was to investigate the effects of three different sperm diluting media, tissue culture medium (TCM‐199), sperm culture medium (Sp‐TALP) and human tubular fluid (HTF) supplemented with varying concentrations of bovine serum albumin (1, 4 and 6%) or polyvinyl alcohol (0.8%) on the acrosomal integrity, motility and viability of canine spermatozoa. Ejaculates collected from four dogs were diluted in all media and spermatozoa were separated from seminal plasma by the swim‐up technique. Sperm progressive motility was assessed using a phase contrast microscope. Viability and acrosomal integrity were evaluated using a dual stain technique (Giemsa–Trypan blue). The results demonstrated that the number of live canine spermatozoa was similar in culture media supplemented or not supplemented with macromolecules. A minimal concentration of albumin (1%) in the three media showed similar effects on vitality, motility and acrosomal integrity, as had higher concentrations (4 and 6%). The percentage of acrosome‐intact spermatozoa was markedly higher after HTF (94.1%) than after TCM‐199 (70.1%) or Sp‐TALP (71.0%) without supplementation. It is concluded that serum bovine albumin, irrespective of the concentration, preserved sperm viability and function, and HTF is the most suitable medium for preserving the acrosome in canine spermatozoa prepared for in vitro manipulation through short incubation.     

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.     

Reduced Immunolabelling of a Porcine Oocyte Membrane Protein Reflects Reduced Fertilizability of Porcine Oocytes Following Elevated Ambient Temperature

Decreased fertility in pigs is a common occurrence during summer months. An objective of the current experiments was to evaluate if elevated ambient temperature altered the oocyte plasma membrane including potential receptors for sperm. This would potentially contribute to reduced fertilizability. Treated gilts were exposed in vivo to 32°C for 12 h per day and 20°C for the remaining 12 h per day for 7 days; control gilts were exposed to 22°C for 12 h and 20°C for the remaining 12 h each day. Cumulus–oocyte complexes were also aspirated from ovaries obtained from gilts maintained at thermoneutral ambient temperature and matured in vitro at 38.5°C or 40°C. Relative abundance of a porcine oocyte membrane protein was examined by intensity of immunolabelling of the in vivo and in vitro matured oocytes evaluated with confocal microscopy; fertilizability of the in vitro matured oocytes was evaluated in in vitro fertilization assays. Oocytes obtained from gilts exposed to elevated ambient temperature for 7 days had reduced immunolabelling compared with oocytes from control gilts (p < 0.05). Similarly, oocytes matured in vitro for 44 h at elevated ambient temperature had reduced immunolabelling and reduced fertilizability compared with oocytes matured at 38.5°C (p < 0.01 and p < 0.05). These results suggest porcine oocyte quality is reduced by elevated ambient temperature and immunolabelling of oocytes with antibodies to specific membrane proteins may be effective to evaluate some aspects of oocyte quality.     

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.     

Ultrastructure changes in buffalo (Bubalus bubalis) oocytes before and after maturation in vitro with sericin

The aim of this research was to identify the changes in the cytoplasmic ultrastructure of immature and matured oocytes in buffalo (Bubalus bubalis ). Oocytes were matured in vitro in tissue culture medium?199 with and without sericin, and then analyzed by light and transmission electron microscopy. The experiment result showed that the nuclear maturation rate of buffalo oocytes was significantly higher in the presence of sericin (80.6%) than without sericin (68.1%) (P  < 0.05). The immature oocytes were characterized by cortical granule clusters in the ooplasm and the absence of perivitelline space (PVS ). In contrast, the oocytes matured either with or without sericin showed the formation of PVS , erected microvilli, the migration of cortical granules to the cytoplasmic periphery, and the clear appearance of the mitochondria and vesicle in the oolemma. Interestingly, matured oocytes with sericin have smaller cortical granules than do immature oocytes (P  < 0.05). In conclusion, supplementation of 0.05% sericin in the maturation medium can enhance the maturation rate of buffalo oocytes. Several cytoplasmic ultrastructures were relocated and modulated during the in vitro maturation process of buffalo oocytes: PVS development, cortical granules migration to periphery, and mitochondria and vesicles in the cortical region. The ultrastructure was similar between the groups with and without sericin.     

Efficiency of three boar sperm enrichment techniques

The objective of the study was to investigate the efficiency of three enrichment methods to separate boar spermatozoa. Twenty-four ejaculates from 12 boars (2 ejaculates/boar) were extended (30 × 10⁶ spermatozoa/mL) in commercial Beltsville Thawing Solution. Each semen sample was processed with glass wool column (GW) and glass beads (GB) filtration and with the single-layer centrifugation (SLC) technique. Semen samples before (control; C) and after treatment were evaluated for sperm CASA motility/kinetics and concentration, viability, morphology and chromatin integrity. Data were analysed with mixed models. The concentration of total and motile spermatozoa was significantly decreased after treatment in groups GW and SLC, but not in group GB. Group GW showed increased values of WOB compared with both groups C and GB. Group GB showed greater values of rapid movement spermatozoa and lower values of slow movement spermatozoa compared with group C. In group SLC, higher values of VSL, LIN and STR were observed compared with group C. In conclusion, all techniques under examination enhanced various CASA variables. Based on our results, the GB method is a promising alternative separation technique for boar sperm and deserves further research regarding swine in vitro fertilization.     

Development of Procedures for Sex-sorting Frozen–Thawed Bovine Spermatozoa

Dairy bull sperm may be sex‐sorted, frozen and used to artificially inseminate heifers with acceptable fertility if the herd is well‐managed. One drawback to the technology is that donor bulls must be located within a short distance of the sorting facility in order to collect semen, which limits the number of bulls from which sorted sperm are available. A successful method used to overcome this limitation in sheep is sex‐sorting from frozen–thawed semen and refreezing for artificial insemination. This technique is attractive to the dairy industry, and therefore a series of three experiments was designed to investigate the optimal methods to prepare, sex‐sort and re‐freeze frozen–thawed bovine sperm. Sperm were prepared for sorting by density gradient separation in either PureSperm^® or BoviPure?, followed by staining in one of three diluents (Androhep^®, Bovine Sheath Fluid + 0.3% BSA or TALP buffer). Sperm were sorted and collected into Test yolk buffer, and frozen in an extender containing 0, 0.25, 0.375 or 0.5% Equex STM Paste. Frozen–thawed sperm were better orientated (p = 0.006) and had fewer damaged membranes (8.7 ± 0.6% vs 19.5 ± 2.4%; p = 0.003) after centrifugation in PureSperm^® rather than BoviPure? gradients. Sperm orientation (p < 0.05) and motility (69.9 ± 3.0 vs 55.6 ± 4.0; p < 0.001) were highest after staining in Androhep^® rather than in TALP buffer. Sperm were more motile (58.2 ± 4.7 vs 38.7 ± 3.5; p < 0.001) and had better acrosome integrity (74.3 ± 2.9 vs 66.8 ± 2.0; p < 0.001) after freezing in an extender containing 0.375% Equex STM Paste than in extender without Equex. Hence, a protocol has been developed to allow frozen–thawed bull sperm to be sex‐sorted with high resolution between the sexes, then re‐frozen and thawed with retention of motility and acrosome integrity.     

Phosphofructokinase and Malate Dehydrogenase Participate in the In Vitro Maturation of Porcine Oocytes

Oocyte maturation depends on the metabolic activity of cumulus–oocyte complex (COC) that performs nutritive and regulatory functions during this process. In this work, the enzymes [phosphofructokinase (PFK) and malate dehydrogenase (MDH)] were tested to elucidate the metabolic profile of porcine COCs during the in vitro maturation (IVM). Enzymatic activity was expressed in U/COC and U/mg protein (specific activity) as mean ± SEM. In vitro maturation was performed with 2‐oxoglutarate (5, 10 and 20 mm ) or hydroxymalonate (30, 60 and 100 mm ) inhibitors of PFK and MDH, respectively. The PFK and MDH activities (U) remained constant during maturation. For PFK, the U were (2.48 ± 0.23) 10^?5 and (2.54 ± 0.32) 10^?5, and for MDH, the U were (4.72 ± 0.42) 10^?5 and (4.38 ± 0.25) 10^?5 for immature and in vitro matured COCs, respectively. The specific activities were significantly lower after IVM, for PFK (4.29 ± 0.48) 10^?3 and (0.94 ± 0.12) 10^?3, and for MDH (9.08 ± 0.93) 10^?3 and (1.89 ± 0.10) 10^?3 for immature and in vitro matured COCs, respectively. In vitro maturation percentages and enzymatic activity diminished with 20 mm 2‐oxoglutarate or 60 mm hydroxymalonate (p < 0.05). Viability was not affected by any concentration of the inhibitors evaluated. The U remained unchanged during IVM; however, the increase in the total protein content per COC provoked a decrease in the specific activity of both enzymes. Phosphofructokinase and MDH necessary for oocyte IVM would be already present in the immature oocyte. The presence of inhibitors of these enzymes impairs the meiotic maturation. Therefore, the participation of these enzymes in the energy metabolism of the porcine oocyte during IVM is confirmed in this study.     

Relation between respiratory activity and sperm parameters in boar spermatozoa cryopreserved with alpha‐tocopherol and selected by Sephadex

Our aim was to evaluate the effect of Sephadex filtration on respiratory activity of porcine spermatozoa and its relation with quality and functional sperm parameters. Samples were evaluated regarding oxygen uptake and sperm parameters: motility, plasma and acrosome membrane integrity, capacitation and acrosome reaction induction in vitro, plasma membrane functionality, determined by the hypo‐osmotic swelling test (HOST), and lipid peroxidation assessed by thiobarbituric acid assay. Sephadex filtration improved all routine quality parameters (motility, plasma and acrosome membrane integrity) and functional parameters (HOST, in vitro capacitation and true acrosome reaction levels) and produced a significant decrease in cryocapacitation and lipid peroxidation. Oxygen uptake increased in Sephadex samples (41 ± 7%) respect to single washing. Oxygen addition of carbonyl‐cyanide‐m‐chlorophenylhydrazone (CCCP) confirmed mitochondrial coupling in washed and Sephadex samples; showing an increase of 2.6 and 4.2 times for oxygen consumption in single washing and Sephadex ones, respectively. The increase in oxygen uptake with succinate addition with respect to basal oxygen uptake was significantly lower in Sephadex samples (63 ± 25%) than in the washed ones (183 ± 35%). Sephadex samples showed higher mitochondrial activity measured by oxygen consumption and improved quality and functional parameters. Our study recommends this protocol due to the fact that this filtration method removes dead or damaged spermatozoa allowing to obtain cryopreserved boar spermatozoa with optimized fertilizing capacity.