Estrategias de conservación de semen de morueco en función del origen espermático

184 p.En la presente tesis doctoral realizamos una serie de experiencias encaminadas a mejorar la viabilidad y eficacia de los bancos de germoplasma en la especie ovina a partir de la adaptación especifica de los protocolos estándar de criopreservación espermátic

Effect of storage method and extender osmolality in the quality of cryopreserved epididymal ram spermatozoa

P. 188-199Post-mortem sperm recovery and cryopreservation could be a complement to germplasm banking in sheep, especially for endangered breeds. This study is an attempt to identify factors for improving the success of cryopreserving ram epididymal spermatozoa, considering the decrease of sperm quality with post-mortem time. Epididymal spermatozoa from 9 rams were kept at 5 °C using three storage methods: within the epididymes, undiluted sperm mass, and diluted in extenders of different osmolality (TES–Tris–fructose at 320, 370 or 420 mOsm/kg, 20% egg yolk, 8% glycerol). At 0, 24, 48 and 72 h, spermatozoa were cryopreserved using each extender. Samples were analyzed before and after cryopreservation by CASA (motility) and flow cytometry (viability and acrosomal status). Post-mortem time decreased pre-freezing and post-thawing sperm quality. Some storage × extender combinations improved the effect of post-mortem time on sperm quality. Both epididymis storage combined with the 420 extender, and storing the spermatozoa diluted in the 320 extender improved post-thawing quality, especially at long post-mortem times. Storing the spermatozoa diluted in the 370 extender was detrimental for the acrosomal status. These findings have practical applications. The simplest storage method (within the epididymes) seems to be adequate if hyperosmotic extenders were used for freezing. An alternative method could be storing the spermatozoa diluted in a hypoosmotic extender. These recommendations are limited to the osmolalities tested in this study (420 mOsm/kg and 320 mOsm/kg); other osmolalities should be tested.S

Post-thawing quality and incubation resilience of cryopreserved ram spermatozoa are affected by antioxidant supplementation and choice of extender

P. 520-528The performance of cryopreserved semen in ovine artificial insemination still needs improvement. Some antioxidants have been tested, with variable success. We cryopreserved semen from Churra rams using TES-Tris-fructose with 4% glycerol and 10% egg yolk (EY) or 3.5% soybean lecithin (SL), with 1 mM of reduced glutathione (GSH), Trolox, crocin, or cysteamine. Samples were analyzed after thawing and incubation (6 hours, 38 °C) for motility (computer-assisted sperm analysis [CASA]), viability, acrosomal integrity, apoptosis, mitochondrial activity, chromatin status, and lipoperoxidation (malondialdehyde production). Interactions (antioxidant/extender/incubation) were significant for most variables. Extenders yielded similar results, although SL depressed mitochondrial activity and linearity (P < 0.001), it improved motility (P < 0.05), DNA fragmentation (P < 0.05), and acrosomal damage (P < 0.001). The control, GSH, and Trolox showed greater viability with SL (P < 0.01). Cysteamine depressed motility (0 hours: 51.6 ± 2.0% vs. 32.3 ± 4.3%; 6 hours: no motility vs. 32.5 ± 1.9%; P < 0.001), but improved viability when using EY (P = 0.004). Crocin increased acrosomal damage (P = 0.022) but improved linearity-related parameters after thawing (P = 0.014). Trolox considerably reduced malondialdehyde production in both extenders (8.6 ± 0.4 nmol per 108 cells vs. 14.2 ± 0.3 in EY and 20 ± 0.6 in SL; P < 0.001). Interestingly, thiol antioxidants (cysteamine and GSH) increased DNA fragmentation (percentage of DNA fragmentation index), whereas crocin reduced it (P < 0.05). Interactions between extender and antioxidant must be taken into account for improving sperm cryopreservation. Soybean lecithin seems to be a suitable replacement for EY, but its effect on mitochondria must be investigated. Trolox and crocin might be useful for ram semen freezing.S

Undiluted or extended storage of ram epididymal spermatozoa as alternatives to refrigerating the whole epididymes

P. 76-82The effect of storage procedure at 5 °C on the quality of ram spermatozoa from the cauda epididymis was analyzed. Two strategies were tested at 0, 24, 48 and 72 h post-mortem: (1) spermatozoa held in the epididymal fluid and stored either in the cauda epididymis (In-EPID) or in vitro (Ex-EPID), (2) epididymal spermatozoa extended in three media at 320, 370 and 420 mOsm/kg (D320, D370, D420). Analyzed parameters were: osmolality, pH, motility, acrosomal status and viability. In experiment 1, osmolality of the In-EPID samples, but not in Ex-EPID, increased with post-mortem time. Motility of In-EPID spermatozoa in samples, after 24 h post-mortem, was higher compared to the Ex-EPID samples, although differences decreased at 48 and 72 h. In experiment 2, total (TM) and progressive motility (PM) were not significantly affected by storage time for D320 and In-EPID samples. However, the motility of D370 and D420 samples significantly decreased with time. TM and PM of D320 were significantly higher than D370 and D420 at 72 h. At 24 h, sperm viability was higher for In-EPID (80.7 ± 3.4%) than for the extended samples (44.8 ± 2.9%, 37.7 ± 3.9% and 48.6 ± 6.0% for D320, D370 and D420, respectively), which also decreased faster with time. At 24 h, the percentage of damaged acrosomes was low and similar for the four methods of storage, but damaged acrosomes increased with time for D320 and D370. Storing the spermatozoa in the epididymis is a good strategy for maintaining sperm quality in ram, at least for 48 h. The D320 extender preserve motility of epididymal spermatozoa but does not protect the status of the acrosome.S

Sperm concentration at freezing affects post-thaw quality and fertility of ram semen

P. 1111-1118We have investigated the effect of sperm concentration in the freezing doses 200, 400, 800, and 1600 × 106 mL−1 on the post-thaw quality and fertility of ram semen. Semen was collected from seven adult Churra rams by artificial vagina during the breeding season. The semen was diluted in an extender (TES-Tris-fructose, 20% egg yolk, and 4% glycerol), to a final concentration of 200, 400, 800, or 1600 × 106 mL−1 and frozen. Doses were analyzed post-thawing for motility (computer-assisted sperm analysis system [CASA]), viability, and acrosomal status (fluorescence probes propidium iodide [PI]/peanut agglutinin conjugated with fluorescein thiocyanate (PNA-FITC), SYBR-14/PI [Invitrogen; Barcelona, Spain] and YO-PRO-1/PI [Invitrogen; Barcelona, Spain]). Total motility and velocity were lower for 1600 × 106 mL−1 doses, while progressive motility and viability were lower both for 800 and 1600 × 106 mL−1. The proportion of viable spermatozoa showing increased membrane permeability (YO-PRO-1+) rose in 800 and 1200 × 106 mL−1. Intrauterine inseminations were performed with the 200, 400, and 800 × 106 mL−1 doses at a fixed sperm number (25 × 106 per uterine horn) in synchronized ewes. Fertility (lambing rate) was similar for semen frozen at 200 (57.5%) or 400 × 106 mL−1 (54.4%), whereas it was significantly lower for 800 × 106 mL−1 (45.5%). In conclusion, increasing sperm concentration in cryopreserved semen, at least at 800 × 106 mL−1 and more, adversely affects the postthawing quality and fertility of ram semen.S

Refrigerated storage of ram sperm in presence of Trolox and GSH antioxidants: Effect of temperature, extender and storage time

P. 137-147Antioxidants have a potential to improve the quality and fertility of refrigerated-stored ram semen. Reduced glutathione (GSH) and Trolox (0.2, 1 and 5 mM) were evaluated in ram semen preserved at 15 and 5 °C up to 48 and 96 h, respectively. Extenders were also evaluated (15 °C: Tris-citrate-fructose, TCF, without lipids, and TES-Tris-fructose 10% egg yolk, TTF-EY; 5 °C: TTF-EY and 3.5% soybean lecithin, TTF-SL; INRA96 at both temperatures). Storage at 5 °C resulted in poorer quality than 15 °C up to 48 h, while allowing acceptable quality at 96 h. Antioxidants had few effects on sperm quality, with use of Trolox resulting in reduced motility and viability in TCF. Storage at 15 °C in the TCF extender resulted in decreased motility, viability and mitochondrial activity compared with use of TTF-EY. Sperm quality when storage was at 5 °C was similar, but storage in TTF-SL resulted in decreased motility and mitochondrial activity. Acrosomal status was only slightly affected by extender and antioxidant. Mitochondrial activity was improved by antioxidants in TTF-SL, and GSH at 5 mM when semen was stored at 5 °C in TTF-EY. A preliminary artificial insemination trial indicated that supplementation with GSH has the potential for improving lambing (P < 0.1). In conclusion, use of antioxidants resulted in lesser effects than extender composition or storage time on quality of ram semen. Use of Trolox negatively impacted sperm quality and GSH had some positive impacts. The use of soybean lecithin requires further research to assess its impact on mitochondria.S

Specificity of the extender used for freezing ram sperm depends of the spermatozoa source (ejaculate, electroejaculate or epididymis)

P. 145-154The objective of this study was to identify possible specificity in the extender formulation for the cryopreservation of ram spermatozoa recovered from three origins (ejaculate, electroejaculate or epididymis), by evaluating post-thawing sperm quality and fertility. Ejaculated, electroejaculated or epididymal spermatozoa samples obtained from identical rams (8) were cryopreserved in four different extenders (TES-Tris–fructose with one of two egg yolk concentrations: 10% Y10 and 20% Y20, and with one of two glycerol rates: 4% G4 and 8% G8). Samples were analyzed before and after cryopreservation by CASA (motility) and flow cytometry (viability with SYBR-14/PI and acrosomal status with PNA/PI). Spermatozoa obtained by electroejaculation were of poorer quality after freezing/thawing, demonstrating that protocols for these samples need to be optimized. Egg yolk at 20% was more appropriate for freezing sperm from any of the sources. In general, 4% glycerol improved the quality of post-thawing samples recovered from ejaculate and electroejaculate, while 8% glycerol was more appropriate for samples recovered from the epididymis. Based on these results, an analysis of fertility was conducted. Fertility rates were similar between ewe groups inseminated with post-thawed sperm obtained from two sources: ejaculate (cryopreserved in Y20 + G4), and cauda epididymis (Y20 + G8), and this rate was less in the electroejaculated sample (Y20 + G4)S

The percentage of spermatozoa lost during the centrifugation of brown bear (Ursus arctos) ejaculates is associated with some spermatozoa quality and seminal plasma characteristics

P. 113-121Cryopreservation of brown bear (Ursus arctos) semen requires centrifugation to increase concentration and/or remove urine contamination. However, a percentage of the spermatozoa are lost in the process. This percentage varies considerably between males and ejaculates, and we have studied the effect of sperm quality and seminal plasma characteristics on the spermatozoa recovery rate after centrifugation. One hundred and thirty one sperm samples obtained from fifteen brown bear males by electroejaculation under general anaesthesia were used. The ejaculates were centrifuged 600 × g for 6 min. Motility was assessed by CASA, and acrosomal status (PNA-FITC) and viability (SYBR-14/propidium iodide) were determined by flow cytometry. Seminal plasma characteristics (albumin, alkaline phosphatase, calcium, cholesterol, creatine, glucose, glutamic oxaloacetic transaminase (GOT), lactate, lipase, magnesium, phosphate and total protein) were determined by a biochemical and gas analysis. Total motility (r = 0.26; P = 0.005) and cell viability (r = 0.20; P = 0.033) were positively correlated with the percentage of recovered spermatozoa. Sperm recovery was correlated with the concentration of several components of seminal plasma: negatively with glucose concentration (r = −0.47; P = 0.005) and positively with the enzymes GOT (r = 0.36; P = 0.040) and lactate dehydrogenase (r = 0.36; P = 0.041). After sorting the data into classes according to sperm recovery (Low: 0–39, Medium: 40–69, High: 70–100), we observed that the samples with a lower recovery rate derived from ejaculates with lower values for TM, VAP and viability (P < 0.05). Multiple regression analysis rendered two models to define the post-centrifugation spermatozoa recovery which included total motility and damaged acrosome or glucose, GOT and lactate dehydrogenase. We discuss these relationships and their implications in the electroejaculation procedure and the handling of the sample during centrifugation.S