Improved cryopreservation protocol for Blanca-Celtibérica buck semen collected by electroejaculation

The collection of sperm samples by electroejaculation (EE) leads to an increase of the production of seminal plasma which could modify the tolerance of spermatozoa to the cryopreservation procedure. This study aims to compare a standard sperm cryopreservation protocol for samples collected by artificial vagina (AV) with the same protocol and modifications to this for samples obtained by EE. Semen from six males of Blanca-Celtibérica goat breed was collected by AV (control) and EE, and three experiments were conducted. In Experiment 1, it was examined the effects of egg yolk concentration contained in freezing extender (0%, 1.5%, 10% and 20% of egg yolk); in Experiment 2, it was evaluated the cooling rate from 30 to 5 °C (fast: 10 min and slow: 90 min) and the temperature of glycerol addition (30 and 5 °C); and in Experiment 3, it was examined the time of equilibration at 5 °C (0, 1, 2 or 3 h). A heterologous in vitro fertilization test was carried out in order to compare the fertility of control samples with that resulting from the EE protocol which showed the highest sperm quality. Results showed greater sperm motility parameters after thawing for control samples cryopreserved in standard conditions in the three experiments. For samples collected by EE, extender with 20% egg yolk, a slow cooling rate and a longer equilibration time (3 h) provided higher sperm quality, and no differences were observed between temperatures of glycerol addition. Samples collected by EE and cryopreserved with the protocol which yielded the best sperm quality after thawing showed higher fertility compared to AV.     

Evaluation of anesthetic protocol for the collection of semen from captive collared peccaries (Tayassu tajacu) by electroejaculation

The objective of this study is to verify and compare the effects of acepromazine–tiletamine–zolazepam and propofol used in anesthetic protocols for semen collection by electroejaculation from captive collared peccaries. Ten sexually mature animals were physically restrained and anesthetized by either intravenous administration of tiletamine–zolazepam (2 mg/kg) after acepromazine premedication, or a propofol dose of 5 mg/kg. The onset of anesthetic recovery was determined by the animals regaining consciousness and attempting to stand. Semen was collected by electroejaculation and evaluated for volume, pH, sperm concentration, progressive motility, morphology, percentage of live cells and functional membrane integrity. Six anesthetized animals with the acepromazine–tiletamine–zolazepam protocol showed erection, but semen could be collected in only four (40%) attempts. Of the animals anesthetized using propofol, nine showed erection, and the ejaculates were collected in eight (80%) attempts. Furthermore, propofol afforded rapid recovery of animals, and ejaculates with enhanced sperm motility and functional membrane integrity as compared with those collected by the other protocol (P < 0.05). In conclusion, use of propofol for anesthetic restraint of collared peccaries enhanced collection of semen by electroejaculation.     

Micro-quantity straw as a carrier for cryopreservation of oligozoospermic semen samples: Effects of storage times and cryoprotectant

Application of an appropriate freezing carrier is crucial for improving post-thaw recovery of oligozoospermic samples. In this study, our purpose is developing a user-friendly, easy handling and close micro-quantity (MQ) straw along with different freezing media, for cryopreservation of oligozoospermic samples. Twenty oligozoospermic semen samples were collected and mixed with glycerol egg yolk citrate (GEYC) or Spermfreeze^® (SPF) medium. The mixture was loaded into MQ straws, sealed and stored in liquid nitrogen (LN) vapor. After freezing, the straws were transferred into cryotube and plunged into LN. Post-thawed sperm parameters including motion characteristics, viability, membrane and DNA integrity were evaluated one and three months after cryopreservation. The post-thawed sperm parameters were significantly reduced in GEYC and SPF medium compared to fresh samples. No statistically significant differences were seen in sperm characteristics between the two storage times (i.e. month 1 vs. month 3). Furthermore, GEYC medium yielded higher motility, viability and membrane integrity compared to SPF at both storage time-points. Sperm DNA integrity was also improved in GEYC group compared to SPF after 1 month of storage. The findings of our study showed that application of MQ straw along with GEYC, as the cryoprotectant, was beneficial for cryopreservation of low count semen samples.     

Use of two anesthetic combinations for semen collection by electroejaculation from captive coatis (Nasua nasua)

The objective was to compare the effects of ketamine-xylazine or tiletamine-zolazepam combinations as anesthetic protocols for captive coatis (Nasua nasua) for semen collection by electroejaculation. Five mature male coatis were physically restrained and then anesthetized by im injections of ketamine (10 mg/kg) plus xylazine (1 mg/kg) or a tiletamine-zolazepam combination (8 mg/kg). For the two combinations, additional quarter-doses of ketamine or the tiletamine-zolazepam combination were administered when necessary. Semen was collected by electroejaculation and immediately evaluated for color, volume, pH, sperm motility, vigor, morphology, acrosomal integrity, and percentage of live cells. Overall, collection of nine ejaculates was attempted from five animals for each treatment. Regardless of the anesthetic combination, all animals developed an erection during each attempt to collect semen. Ejaculates were obtained in all (9 of 9) attempts that used ketamine-xylazine for anesthesia, but in only 3 of 9 attempts (P < 0.05) when tiletamine-zolazepam was used. All ejaculates contained sperm, with no significant differences in semen characteristics between the two anesthetic combinations. Recovery was smooth in all animals. In conclusion, semen collection by electroejaculation in coatis was significantly more successful with the use of a ketamine-xylazine combination than with tiletamine-zolazepam.     

Evaluation of l-glutamine for cryopreservation of boar spermatozoa

The aim of the present study was to evaluate the protective effect of l-glutamine (l-Gln) against cryopreservation injuries on boar sperm. In Experiment 1, l-Gln from 20 to 80 mM was evaluated as a supplement for a standard freezing extender (egg yolk – EY – 20%, and glycerol 3%). No significant improvement (P > 0.05) was obtained for any post-thaw sperm parameter assessed (objective sperm motility – CASA system – and flow cytometric analysis of plasma and acrosomal membrane integrity −SYBR14/PI/PE-PNA− and plasma membrane stability −M540/YoPro1−). In Experiment 2, l-Gln was evaluated as a partial glycerol substitute in the freezing extender. Significant (P < 0.05) enhancement of post-thaw sperm motion parameters was achieved in sperm frozen in the presence of 2% glycerol and 80 mM l-Gln compared to control (3% glycerol). In Experiment 3, l-Gln was evaluated as an EY substitute in the freezing extender, and no functional sperm were recovered after thawing sperm frozen in the presence of l-Gln and the absence of EY. In conclusion, l-Gln has the ability to cryoprotect boar sperm when it is used as a partial glycerol substitute in the freezing extender.     

Pre-freezing equilibration for 22 h improves post-thaw sperm functions in cryopreserved ram semen by reducing cholesterol efflux

Failure of cervical insemination with cryopreserved semen is hindering implementation of AI in sheep in field condition. Here the effect of equilibration time and catalase on post-thaw qualities of ram semen was investigated. Pooled semen was diluted (800 × 10⁶ sperm mL⁻¹) with a TES-Tris-fructose extender with 6% glycerol, 15% egg yolk and supplemented with 0, 50, 100 and 200 U mL⁻¹ catalase and packaged into 0.25 mL straws. In experiment 1, straws were equilibrated at 5 °C either for 3 h in a cold cabinet (E3) or for 10 (E10) and 22 h (E22) inside a refrigerator. In experiment 2, all straws were equilibrated for 22 h inside refrigerator. Straws were frozen at −25 °C min⁻¹ up to −125 °C using a cell freezer and finally plunged into liquid nitrogen. The post-thaw total and rapid motility were higher (P < 0.05) in E22 compared to E3 and E10. Sperm kinetics was comparable between E3 and E22, but lower in E10. Similarly, acrosome integrity, functional membrane integrity, percent high cholesterol (mCHO) and live-high mitochondrial membrane potential (MMP) were higher (P < 0.05) while live-high intracellular calcium and acrosome-reacted sperm were lower in E22 compared to E3 and E10. The percent rapid motile, high mCHO and live-high MMP were significantly (P < 0.05) lower in catalase-treated samples compared to the control, while the membrane integrity was comparable within the groups. In conclusion, pre-freezing equilibration for 22 h compared to 3 or 10 h resulted in higher post-thaw sperm functions while catalase had negative impact on cryopreservation of ram semen.     

Comparison of heat transfer in liquid and slush nitrogen by numerical simulation of cooling rates for French straws used for sperm cryopreservation

Slush nitrogen (SN₂) is a mixture of solid nitrogen and liquid nitrogen, with an average temperature of −207 °C. To investigate whether plunging a French plastic straw (commonly used for sperm cryopreservation) in SN₂ substantially increases cooling rates with respect to liquid nitrogen (LN₂), a numerical simulation of the heat conduction equation with convective boundary condition was used to predict cooling rates. Calculations performed using heat transfer coefficients in the range of film boiling confirmed the main benefit of plunging a straw in slush over LN₂ did not arise from their temperature difference (−207 vs. −196 °C), but rather from an increase in the external heat transfer coefficient. Numerical simulations using high heat transfer (h) coefficients (assumed to prevail in SN₂) suggested that plunging in SN₂ would increase cooling rates of French straw. This increase of cooling rates was attributed to a less or null film boiling responsible for low heat transfer coefficients in liquid nitrogen when the straw is placed in the solid-liquid mixture or slush. In addition, predicted cooling rates of French straws in SN₂ tended to level-off for high h values, suggesting heat transfer was dictated by heat conduction within the liquid filled plastic straw.