Purification of cryopreserved camel spermatozoa following protease-based semen liquefaction by lectin-functionalized DNA-defrag magnetic nanoparticles.

Although incorporating proteases into sperm medium is considered the most effective procedure to eliminate camel semen viscosity, it drastically affects viability, morpho-functional properties and, hence, fertilization potential of spermatozoa. The present work aimed at evaluating adequacy of employing magnetic nanoparticles-based sperm purification technique for eluting impaired and apoptotic camel spermatozoa from cryopreserved semen doses following protease-based semen liquefaction. Thirty cryopreserved semen doses (50 x 106 sperm/straw) representing the following liquefaction treatments: control (untreated), 0.1 mg/ml papain or 5 U/ml bromelain were used (n = 10 straws per treatment). Immediately after thawing (38°C for 40 s), sperm concentration of each straw within treatment was readjusted to 15 x 106 sperm/mL by dilution in PBS (37°C). Sperm physical and cytological properties were then assessed (non-purified semen). Thereafter, each specimen was subjected to lectin-functionalized DNA-defrag magnetic nanoparticles sperm purification, and the same sperm traits were re-evaluated after undergoing purification (purified semen). Sperm DNA fragmentation level within each group, prior to and after magnetic nano-purification, was also determined by fluorescent imaging. The results showed a dramatic improvement (p < .05) in post-thaw motility (%), viability (%), normal sperm (%), intact acrosome (%) and HOST-reacted (%) spermatozoa in protease-liquefied semen following sperm magnetic nano-purification. Additionally, the highest (p < .05) DNA fragmentation level was recorded in all cryopreserved semen groups prior to purification, whereas the lowest (p < .05) was observed in the protease-treated specimens after magnetic nano-purification. These results indicate that protease-based semen liquefaction prior to cryopreservation in conjunction with magnetic nano-purification post-thawing holds potential for reducing the proportion of damaged and dead spermatozoa, hence improving camel sperm fertilization competence.

Enhancing liquid-chilled storage and cryopreservation capacities of ram spermatozoa by supplementing the diluent with different additives.

OBJECTIVE: In the present study, we determined efficiency of incorporating caffeine, melatonin or omega-3 polyunsaturated fatty acid in the diluent on mitigating consequences of (a) liquid chilled- and (b) cryo-storage of ram spermatozoa. METHODS: In the first experiment, ejaculates (n = 30) were collected from 5 adult rams and were pooled, diluted (1:10) with Tris-citric acid (base diluent) and were split into 4 aliquots assigned for: control (untreated), caffeine (0.1 mM), melatonin (0.3 mM) or omega-3 fatty acids (0.3 mM) (T0). The diluted specimens were stored at 4°C for 48 h, during which sperm physical and cytological properties were evaluated along with oxidative stress indices (T24, T48). In the second experiment, 15 ejaculates (3 per male) were pooled, diluted with glycerolized base diluent (4% glycerol, v/v) and were split corresponding to the same previous treatment groups before being processed for cryopreservation. Post-thaw physical and kinematic sperm properties were assessed by a computer-assisted sperm analysis system. RESULTS: The results clarified superiority of both melatonin and omega-3 supplementation on maintaining (p<0.05) sperm properties, while reducing (p<0.05) lipid peroxidase reaction and enzymatic activities of alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase in preservation medium, compared to caffeine either during liquid-chilled storage or cryopreservation of spermatozoa. CONCLUSION: Melatonin and omega-3 are regarded efficient alternatives to caffeine when processing ram spermatozoa for application of artificial insemination or in vitro fertilization.

Influence of omega-3 incorporation in sperm preservation medium on physical and kinematic properties of chilled and cryopreserved ram spermatozoa.

Two experiments were carried out to investigate the efficiency of supplementing sperm preservation medium with omega-3 polyunsaturated fatty acids on improving liquid-chilled storage and cryopreservation capacity of ram spermatozoa. Ejaculates (n = 100) were collected from five adult rams, Ovis aries, by an artificial vagina twice weekly throughout the period February-April, 2017. After initial evaluation, ejaculates of each collection session from the same males were pooled, diluted (1:10) with Tris-citric acid egg yolk extender, and were further split into five aliquots using a split-sample technique. The first aliquot served as control (omega-free), whereas the other four portions were supplemented with 0.1, 0.2, 0.3 or 0.4 mM omega-3, respectively (T0 ). Thereafter, the diluted specimens were stored at 4°C for 48 hr, during which sperm physical and morphometric properties were evaluated along with oxidative stress indices (T24 , T48 ). Omega-3 levels that efficiently mitigated the detrimental effects of chilled preservation, and maintained preservation aptitude of spermatozoa were further investigated for sperm cryosurvival against control (untreated). Post-thaw physical and kinematic properties of spermatozoa, in all groups, were objectively evaluated by a computer-assisted sperm analysis (CASA) system. The results showed that, at 48 hr of chilled storage, supplementing preservation medium with 0.4 mM omega-3 was positively correlated (p < 0.01) with each of progressive motility, live sperm, intact acrosome and intact cell membrane (r = 0.83, 0.85, 0.85, 0.89, respectively). Furthermore, a positive correlation (p < 0.01) was observed between inclusion of omega-3 in cryopreservation medium and each of post-thaw total sperm motility, progressive motility, live sperm, normal sperm, intact acrosome, intact cell membrane, VCL, VSL, VAP, ALH and STR (r = 0.76, 0.84, 0.79, 0.90, 0.89, 0.91, 0.61, 0.73, 0.65, 0.78 and 0.60, respectively). These results accentuate efficiency of supplementing the diluent with omega-3 fatty acids on improving chilled and cryopreservation aptitude of ram spermatozoa.

Physical and kinematic properties of cryopreserved camel sperm after elimination of semen viscosity by different techniques.

This investigation aimed to determine the influence of using different techniques for liquefaction of semen on post-thaw physical and dynamic characteristics of camel spermatozoa. A total of 144 ejaculates were collected from 3 adult camels, Camelus dromedarius, twice-weekly over 3 consecutive breeding seasons. A raw aliquot of each ejaculate was evaluated for physical and morphological properties, whereas the remaining portion was diluted (1:3) with glycerolated Tris lactose egg yolk extender, and was further subjected to one of the following liquefaction treatments: control (untreated), 5µl/ml α-amylase, 0.1mg/ml papain, 5u/ml bromelain, or 40-kHz nominal ultrasound frequency. The post-thaw objective assessment of cryopreserved spermatozoa, in all groups, was performed by a computer-assisted sperm analysis (CASA) system. The results revealed that all liquefaction treatments improved (P<0.05) post-thaw motility, viability and sperm motion criteria. However, an adverse effect (P<0.05) was observed in acrosome integrity, sperm cell membrane integrity and percent of normal sperm in all enzymatically-treated specimens compared to both control and ultrasound-treated semen. These results elucidate the efficiency of utilizing ultrasound technology for viscosity elimination of camel semen. In addition, developing enzymatic semen liquefaction techniques is imperious to benefit from when applying assisted reproductive technologies, particularly AI and IVF, in camels.

Fixed-time induction of ovulation in camels superovulated by different eCG modalities during the transition period in Egypt : Superovulation in camels during the transition period.

The current investigation aimed to establish a fixed-time induction of ovulation/ insemination protocol in camels superovulated by different equine chorionic gonadotropin (eCG) regimens during the transition period in Egypt (mid-October to mid-November). Seventeen pluriparous camels, Camelus dromedarius, were used. All females retained controlled intra-vaginal drug releasers (CIDRs) for 13 consecutive days, and at CIDR withdrawal, the camels were randomly divided into three groups. The control group (n = 5) received 1 ml saline intra-muscularly (i.m.), whereas remaining camels were superovulated by 2500 IU eCG either in a single shot (SS, n = 6) or in serial decreasing doses over 3 days (DD, n = 6). Ovarian dynamics were monitored by transrectal ultrasonography at 2-day intervals, and ovulation was induced by 5000 IU hCG i.m. The changes in reproductive hormones throughout the period of the study were determined. The results showed that mean values of total no. of follicles and size of dominant follicles remained low (P < 0.05) in all groups until day of CIDR removal. Thereafter, total follicle no. increased (P < 0.05) in both superovulated groups compared to the control, where the dominant follicles attained the highest (P < 0.05) diameter 12 days after the eCG treatment. Double-ovulation rate was higher (P < 0.05) in SS (50%) and DD (66.6%) groups compared to that of control (0.0%). However, 33.3% of the SS group developed large anovulatory follicles (ø > 25 mm), which did not respond to induction to ovulation. These results elucidate that eCG administration in serial decreasing doses generates a reliable superovulatory response in camels, and ovulation can be blindly induced 12 days after the gonadotropin treatment. This fixed-time hormonal protocol represents a sufficient alternative to conventional day-to-day ultrasonography and would have profound implication for enhanced fertility in dromedary camels by facilitating infield application of embryo transfer technique.