Evaluation of epididymis storage temperature and cryopreservation conditions for improved mitochondrial membrane potential,membrane integrity,sperm motility and in vitro fertilization in bovine epididymal sperm

The maintaining of the epididymis at lower temperatures during storage and transport improves sperm quality. Our study aimed to test whether epididymis storage temperature (post‐mortem) and sperm cryopreservation affect sperm kinetics, membrane integrity, mitochondrial potential and fertility capacity. Thirty‐six epididymides were collected from 18 bulls after slaughter and divided into two groups: at 4 or 34°C for 2–3 hr. The sperm was collected from the epididymis cauda. The evaluation consisted of computer‐assisted sperm analysis (CASA), SYBR14/PI/JC1 to evaluate membrane integrity, mitochondrial membrane potential (MMP) and measurement of lipid peroxidation (TBARS). The sperm was then frozen using an automatic device. After thawing, sperm samples were evaluated by the same variables and further in vitro fertilization rates. Cryopreservation negatively affected sperm motility in samples stored at 4 and 34°C. Nevertheless, the 4°C samples yielded higher rates of blastocyst formation. Pre‐freeze sperm motility, progressive motility and velocity were higher in sperm from epididymis stored at 4°C while post‐thaw sperm motility, progressive motility and velocity remained the same among samples from epididymis stored at 4 or 34°C. However, with regard to the kinetic patterns, samples collected from epididymis stored at 34°C had lower values when compared to those stored at 4°C prior the cryopreservation process. Our results indicate that epididymis handling conditions after cryopreservation may affect sperm quality after thawing, especially due to compromised MMP in sperm collected from epididymis stored at higher temperatures.     

Effect of Foetal Bovine Serum on sperm motility,acrosome reaction and spermatic interaction to zona pellucida in alpacas (Vicugna pacos)

The use of foetal bovine serum (FBS) in cell culture media is quite common. However, little is known about the effect of FBS on sperm. The severe difficulties in alpaca reproduction demand the search of new methods for in vitro reproductive management. In the present study, we use for the first time FBS as a supplement in the culture medium for sperm in alpaca, and the effect of FBS on motility, acrosome reaction and sperm binding to the zona pellucida in this species was evaluated. A concentration of 10% v/v FBS was used. The sperm motility with FBS at the first hour was 32.8% (vs. control = 30.0%), whereas at the second hour sperm motility with FBS was 30.2% (vs. control = 28.8%). The acrosome reaction reached an average of 44.0% for treatment with FBS (vs. control = 30.1%). The sperm‐zona pellucida binding assay showed that the samples incubated with FBS had an average of 2.7 bound sperm (vs. control = 1.7). Only a significant difference was observed for sperm motility at the first hour and for the acrosome reaction. It is concluded that FBS favours the capacitation of sperm in alpaca.     

Application of laser-assisted zona drilling to in vitro fertilization of cryopreserved mouse oocytes with spermatozoa from a subfertile transgenic mouse

Development of assisted reproductive technologies is necessary to obtain fertilized oocytes in a subfertile transgenic mouse strain. Here, we showed the application of laser-assisted drilling of the zona pellucida to in vitro fertilization of cryopreserved mouse oocytes with sperm from subfertile transgenic mice (C57BL/6N-Tg(UCP/FAD2)U8 strain). After cryopreservation by vitrification, the recovery and survival rates of the zona-drilled mouse oocytes were 97% (97/100) and 94% (91/97), respectively. In vitro fertilization of the cryopreserved zona-drilled mouse oocytes with sperm from the subfertile transgenic mice was greatly facilitated (60%, 55/91) compared to that of the cryopreserved zona-intact mouse oocytes (11%, 81/768). In vitro fertilized embryos that developed to the 2-cell stage were again cryopreserved by vitrification, and after warming they were transferred into recipient females. Subsequently, six viable offspring were delivered, and all were confirmed to be transgenic mice. These results indicate that laser-assisted zona drilling of oocytes combined with cryopreservation by vitrification may be a useful approach for large-scale production of in vitro fertilized embryos for managing transgenic mouse strains with reproductive disabilities such as subfertile sperm.     

Glycohistochemistry of the zona pellucida of developing oocytes in the rabbit and hare

Lagomorpha are often used as animal models in reproductive experiments. The aim of the present study was to examine the glycoconjugate modifications occurring mainly in the zona pellucida during oocyte growth in the rabbit and hare, using a battery of lectins combined with sialidase digestion and chemical treatments. This histochemical approach made it possible to identify sulpho- and asulpho-carbohydrates in the terminal and/or subterminal position linked to sialic acid residues. The lectins that stained the zona pellucida of both species most effectively were SBA, PNA and WGA, indicating the presence of beta-D-N-acetylgalactosamine, beta-D-galactosamine and N-acetylglucosamine residues. The differences in the glucidic residue content and in their spatial distribution that depended on the species and stage of follicle development were also detected.     

Antioxidants protect proteins' anchorage to the bilayer by improving plasma membrane integrity of ram spermatozoa during liquid preservation in a soya lecithin‐based diluent

Antioxidants are known to prevent the reactive oxygen species (ROS)‐mediated peroxidative damage to the membrane lipids during hypothermic storage of mammalian spermatozoa. We hypothesized here that ROS also affect the lipid–protein interactions, thereby diminishing the membrane's integrity and proteins' anchorage to the bilayer. Antioxidants prevent these damages by scavenging the ROS. Ejaculates from Patanwadi rams were pooled after subjective evaluation and centrifuged using Percoll^®. Sperm pellet was resuspended in soya lecithin–Tris–fructose diluent (400 × 10⁶ cells/ml) containing either antioxidants (100 IU/ml catalase + 10 mM reduced glutathione) or no antioxidant. Aliquots were chilled to 5°C in a cabinet and stored in a refrigerator at 3–5°C for 72 hr. Sperm motility, viability, lipid peroxidation (LPO) and hypo‐osmotic swelling test (HOST) were performed at 0, 24, 48 and 72 hr. Sperm proteins extracted with 0.5% Triton X‐100 were resolved by SDS‐PAGE and quantified using Quantity One software (Bio‐Rad, USA). The rapid motility, linearity and straight‐line velocity (VSL) were found significantly (p < .05) higher in the antioxidant‐treated group compared to the control at 48 hr of storage. Sperm viability was found comparable between the groups. Higher HOST response and lower LPO were found in the antioxidant‐treated sample compared to the control both at 48 and at 72 hr. Overall, the proteins P1 (106.09 kDa), P2 (87.00 kDa) and P4 (51.14 kDa) were lower (p < .05) in the sperm extract of antioxidant‐treated group compared to the control. The content of P4 (51.14 kDa) in sperm extract was found to increase (p < .05) earlier (48 vs. 72 hr) in the control group compared to the antioxidant‐treated group. Altogether, the results suggested that antioxidants reduced LPO in spermatozoa, resulting in higher sperm motility, plasma membrane integrity and protection of proteins' anchorage to the plasma membrane at 48 and 72 hr of storage.     

Pseudorabies virus, porcine parvovirus, and porcine enterovirus interactions with the zona pellucida of the porcine embryo

Porcine embryos (n = 93) were incubated on cell monolayers that had been previously inoculated with pseudorabies virus, porcine parvovirus (PPV), or each of 2 porcine enteroviruses. After 2, 24, or 48 hours of incubation, the embryos were fixed in glutaraldehyde and examined by electron microscopic procedures. It was found that pseudorabies virus adsorbed to the zona pellucida (ZP) and entered sperm tracks in the ZP. The PPV and both enteroviruses entered pores in the ZP and were associated with sperm that were at or near the outer surface of the ZP. In addition, PPV was seen enmeshed in cellular debris on the outer surface of the ZP. Evidence of a productive viral infection of the blastomeres of the embryos was not found.     

Variability in plasma membrane integrity, mitochondrial membrane potential and acrosomal status before and after cryopreservation of bull sperm

In the present study variability of bull sperm concerning percentages of sperm with intact plasma membranes (PMI), high mitochondrial membrane potential (HMMP) and positive acrosomal status (PAS) before and after cryopreservation (vKK; nKK) between bulls and between ejaculates within bulls was examined. Studies were performed on 4 semen samples each of 20 Deutsche Fleckvieh bulls. VKK-Values were 76.5% +/- 9.6% (PMI) 68.3% +/- 8.9% (HMMP) and 9.8 +/- 5.1% (PAS) and nKK-values were 38.1 +/- 14.0% (PMI), 38.2 +/- 14.0% (HMMP) and 30.9 +/- 12.1% (PAS). After freezing, variabilities in sperm parameter values between bulls (nKK: PMI: 49.8%, HMMP 52.1% and PAS: 56.6%) were nearly quite as high or higher than variabilities between ejaculates (nKK: 50.2%, 47.9% and 43.4%). VKK-values of PMI, HMMP and PAS were only fairly to moderately related (0.36 < r < 0.53; P < 0.05) to nKK-values. The results show that PMI, HMMP and PAS did not only vary between bulls, but also between ejaculates within bulls. As there are no high relationships in these sperm parameters between times before and after cryopreservation, each ejaculate should be examined after cryopreservation in order to receive a reliable information about the quality of cryopreserved sperm.     

Effect in vitro of bovine viral diarrhea virus on bovine embryos with the zona pellucida intact,damaged and removed

The in vitro effect of bovine viral diarrhea virus (BVDV) on the survival of day 7 to day 7.5 bovine embryos collected from superovulated donors was studied. Fifty-four experimental embryos with the zona pellucida (ZP) intact, damaged or removed were exposed to 1×10⁴ TCD₅₀/ml of the NADL cytopathic strain of BVDC at 37°C for 24 hrs and compared to 36 control embryos that were cultured for 24 hr. Seven embryos with the ZP-removed were similarly exposed for 48 hrs and compared to five control embryos. The overall survival rate was 68% for embryos exposed to BVDV for 24 hrs and 77% for embryos not exposed (P>0.05). Extended exposure of the embryos with the ZP removed to virus for 48 hrs did not affect their survival rate compared to controls. Damage to the ZP by cracking or total removal of the ZP by micromanipulation or acidic Tyrode's solution had no effect on subsequent embryonic survival in the presence of BVDV. It was concluded that exposure to BVDV in vitro is not cytopathic for morula and blastocyst stage bovine embryos over a 48 hr period, even when they are not protected by the ZP.     

Adaptation of protein oxidation to protein intake in the domestic cat

It is well-accepted that cats require more dietary protein than omnivores and herbivores. Work on hepatic enzyme activities showed that cats lack the ability to regulate the urea cycle enzymes in response to the dietary supply of protein. It was thus hypothesized that the high protein requirement of cats is due to an inability to regulate these enzymes, limiting adaptation to a low protein diet. We used indirect respiration calorimetry to assess the in vivo ability of cats to adapt substrate oxidation to different levels of dietary protein, including one below their protein requirement. In random order, eight cats consumed each of four semi-purified diets containing 7.7% (LP), 14.6% (AP), 27.3% (MP) and 51.1% (HP) of ME from protein. Cats consumed each diet for at least 14 days and then completed a 5-day nitrogen balance trial and at least 2, 12-hour indirect calorimetry measurements. The data were analyzed by anova using the Mixed procedure of SAS and are expressed as mean ± SEM. There was a significant effect of diet on protein oxidation (p < 0.0001), measuring 9.8 ± 0.5%, 13.4 ± 0.9%, 23.5 ± 0.8% and 49.0 ± 1.8% of total energy expenditure on the LP, AP, MP and HP diets, respectively. The ratio of protein oxidation/protein intake was significantly higher with the LP diet (1.27 ± 0.07) than the other three diets (AP, 0.92 ± 0.06; MP, 0.86 ± 0.03; HP, 0.96 ± 0.04; p < 0.0001), indicating a net loss of protein on the LP diet. Thus, cats adapted to a wide range of dietary protein concentrations, but were unable to fully adapt to the LP diet.     

Spermatic and oxidative profile of domestic cat (Felis catus) epididymal sperm subjected to different cooling times (24, 48 and 72 hours)

Cooling stored epididymal samples for several days allows facilities to transport and process genetic material post‐mortem. Improvements to this practice allow the preservation of sperm from domestic cats, which are the ideal study model for wild felids. However, the modifications in spermatic features and the oxidative profile are not fully understood in cats. This information is necessary for the development of biotechniques, such as new extenders for cryopreservation. Therefore, the purpose of this study was to evaluate the spermatic and oxidative profile in samples from the epididymal cauda of domestic cats cooled at 5°C for 24, 48 and 72 hr. Spermatozoa were collected from the epididymis cauda. Evaluations consisted of computer‐assisted sperm analysis (CASA), plasma membrane integrity (eosin/nigrosin), acrosome integrity (fast green/rose bengal), sperm morphology, sperm DNA integrity (toluidine blue), mitochondrial activity (3′3 diaminobenzidine), activity of the antioxidant enzymes glutathione peroxidase (GPx) and superoxide dismutase (SOD), measurement of lipid peroxidation (TBARS) and protein oxidation. A decrease in sperm motility parameters was observed after 72 hr of cooling (i.e. total and progressive) with a higher percentage of minor (37.7 ± 6.3%) and total defects (53.4 ± 6.3%). Additionally, a decrease in high mitochondrial activity (Class I: 16.6 ± 2.2%) occurred after 72 hr. The decrease in motility rates after a long cooling time probably was caused by the increase in sperm abnormalities. A long cooling time causes cold shock and mitochondrial exhaustion, but there was no observed change with the oxidative stress condition. Therefore, cat epididymal sperm stored at 5°C appear to maintain a high quality for up to 48 hr of cooling time.     

Adherence of bovine viral diarrhea virus to bovine oocytes and embryos with a hardened zona pellucida cultured in vitro

The purpose of this study was to investigate the adherence of bovine viral diarrhea virus (BVDV) to bovine mature, or immature, cumulus-free oocytes and to in vitro fertilized embryos, maintained in vitro in a ligated bovine oviduct to allow for the hardening of the zona pellucida. Incubation of the oocytes and embryos in the oviduct for 5 h caused hardening of the zona pellucida as measured by resistance to pronase digestion (which increased from approximately 3 min to 7 h; P >0.001). However, there was no difference between the number of infected oocytes and embryos (n = 965 in 193 samples) following experimental exposure to BVDV regardless of whether or not they were previously incubated in the oviduct (P > 0.05). It was concluded that the modification of the proteolytic resistance properties of the zona pellucida during in vitro oviductal incubation did not influence the adherence of BVDV to zona pellucida of oocytes or in vitro fertilized embryos.     

Association between embryonic loss and damage to the zona pellucida by invasive micromanipulation during oviductal transfer of early-stage embryos in pigs

The aim of this study was to examine the impact of zona pellucida damage, which might arise during somatic cell nuclear transfer (SCNT), on the development and survival of transferred embryos. The zonae pellucidae of in vitro matured oocytes were either punctured with 8- to 10-microm square-ended nuclear injection pipettes and piezo pulses or slit with 35- to 40-microm enucleation pipettes. Intact oocytes were used as controls. These oocytes were electroactivated to induce parthenogenesis and transferred to the oviducts of estrus-synchronized recipient gilts. After 5 to 7 days, the recipient uteri were flushed to collect embryos, and embryonic development (morula-blastocyst stage embryos/collected embryos) and survival (viable embryos/collected embryos) were determined. In total, 221 zona-punctured, 129 zona-slitted and 57 intact embryos were transplanted into four, two and two gilts, respectively. The efficiency of embryo recovery was similar in all groups (64.3 to 79.1%). However, the zona-penetrated and incised embryos exhibited unstable development and survival compared with the controls; development and survival of the control embryos were 94.7 and 87.7%, whereas those of the zona-punctured embryos were 69.0 and 47.9% (P<0.01) and those of the zona-slit embryos were 64.7 and 50.0% (P<0.01). Cells with large foci that appeared to be macrophage giant cells were observed at the surface or inside the degenerated zona-damaged embryos. These results indicate that the recipient's immune response to damage to the zona pellucida may impair embryonic development after transplantation to the oviduct. This may be one of the factors causing the reduced efficiency of live progeny production by SCNT.     

Sperm motility parameters to evaluate the seminal quality of Boa constrictor occidentalis, a threatened snake species

Semen quality analysis constitutes a powerful tool to evaluate the fertility potential of males in threatened species. The Argentine boa constrictor or lampalagua (Boa constrictor occidentalis) is a threatened snake species and has been included in Appendix I of CITES. The objective of this work is to characterize the sperm of B. c. occidentalis on the bases of dynamic parameters to improve this species conservation. Dynamic parameters were measured in sperm samples using videomicroscopy and image analysis software. The sperm population showed a high degree of heterogeneity in velocity parameter values and 95% of the cells showed a linear pattern of movement. Studies in other species indicate that the number of motile spermatozoa and their movement speed is directly correlated with fertilization success. This work will help to establish basic parameter values for the evaluation of the reproductive potential of populations of B. c. occidentalis and to resolve questions referred to its reproductive strategies.