Article

Tris–Egg Yolk–Glycerol (TEY) Extender Developed for Freezing Dog Semen is a Good Option to Cryopreserve Bovine Epididymal Sperm Cells

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Abstract

ContentsCryopreservation of epididymal spermatozoa is often performed after shipping the excised testis–epididymis complexes, under refrigeration, to a specialized laboratory. However, epididymal spermatozoa can be collected immediately after excision of the epididymis and sent extended and refrigerated to a laboratory for cryopreservation. In this experiment, we evaluated the effect of both methods of cold storage bovine epididymal spermatozoa as well as of two different extenders on spermatozoa characteristics after freeze–thawing. For that, spermatozoa collected from the caudae epididymis of 19 bulls were extended and cryopreserved in either AndroMed® or a Tris–egg yolk (TEY)-based extender. Cryopreservation of sperm cells was performed immediately after castration (Group A, n = 9) or after cold storage for 24 h diluted in the two extenders and (Group B, n = 9) and also after cold storage for 24 h within the whole epididymis (Group C, n = 10). Sperm subjective progressive motility (light microscopy), plasma membrane integrity (hypoosmotic swelling test) and sperm viability (eosin–nigrosin) were evaluated. In vitro fertilization and culture (IVF) was performed to assess the blastocyst rate. No differences (p > 0.05) were observed on post-thaw sperm parameters between samples from Group A, B and C. TEY extended samples presented a higher (p < 0.01) percentage of progressive motile and live sperm, than those extended in AndroMed®. Blastocyst rate after IVF differed only (p < 0.05) between the reference group (IVF performed with frozen semen with known in vitro fertility) and Group A extended in AndroMed®. We conclude that when cryopreservation facilities are distant from the collection site, bovine epididymal sperm can be shipped chilled overnight either within the epididymal tail or after dilution without deleterious effect on post-thaw sperm quality. TEY extender was more suitable for cold storage and freezing bovine epididymal sperm, than the commercial extender AndroMed®.

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... In none of these studies, however, have the spermatozoa been used for in vitro fertilization (IVF), which is relevant for preservation of genetic material and for evaluation of the fertilizing capacity of spermatozoa after in vitro handling. In contast, such studies have been carried out in bovids, but they are few and have resulted in fairly low in vitro embryo blastocyst rates [6% (James, 2004); 13% (Martins et al., 2009); 12% (Chaveiro et al., 2015); and 3-16% (Lopes et al., 2015)]. Furthermore, no particular information has been provided relating to the importance of the male's age with respect to the spermatozoa. ...
... Our results showed that refrigeration followed by cryopreservation of epididymal spermatozoa from both group 1 and group 2 bulls allowed production of embryos in vitro with satisfying rates, especially from group 2 bulls. Other studies have demonstrated lower developmental rates (James, 2004;Martins et al., 2009;Chaveiro et al., 2015;Lopes et al., 2015), and this may be caused by differences in methods for transport and storage, e.g. a 24 h transportation time of the oocytes (James, 2004), different extenders for spermatozoal handling (Lopes et al., 2015) or other freezing and thawing rates (Chaveiro et al., 2015). ...
... Our results showed that refrigeration followed by cryopreservation of epididymal spermatozoa from both group 1 and group 2 bulls allowed production of embryos in vitro with satisfying rates, especially from group 2 bulls. Other studies have demonstrated lower developmental rates (James, 2004;Martins et al., 2009;Chaveiro et al., 2015;Lopes et al., 2015), and this may be caused by differences in methods for transport and storage, e.g. a 24 h transportation time of the oocytes (James, 2004), different extenders for spermatozoal handling (Lopes et al., 2015) or other freezing and thawing rates (Chaveiro et al., 2015). ...
Article
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The aim of this study was to establish and validate a reliable and efficient protocol for the recovery and cryopreservation of epididymal spermatozoa used for in vitro fertilization, using bulls of two different age classes. Testicles from 26 (37–51 weeks old, group 1) and 19 (52–115 weeks old, group 2) Danish Holstein bulls were collected after slaughter and stored at 5°C. After 0, 24 or 48 h, epididymides were isolated and spermatozoa collected. Assessments included spermatozoal motility, viability and morphology before and after cryopreservation and in vitro embryo production. Results showed that live spermatozoa can be collected from epididymides of bulls after their death. Storage of the testicles at 5°C for 24 h followed by cryopreservation of recovered epididymal spermatozoa resulted in 21% (group 1) and 31% (group 2) blastocysts produced in vitro. These results illustrate that epididymal spermatozoa recovered from testicles kept in specific conditions can be used to preserve genetic material from endangered and threatened species or populations in nature as well as in domestic and zoo animals. Cite as: Strand J, Ragborg MM, Pedersen HS, Kristensen TN, Pertoldi C, Callesen H (2016) Effects of post-mortem storage conditions of bovine epididymides on sperm characteristics: investigating a tool for preservation of sperm from endangered species.
... 8 These differences may influence sperm response to cryopreservation and their interaction with semen extenders. Bovine epididymal sperm has been routinely cryopreserved using egg yolkbased extenders, [9][10][11][12][13][14][15][16][17] but studies testing liposome-based extenders for cryopreservation of epididymal sperm are lacking. In addition, most protocols for epididymal sperm freezing were developed for Bos indicus or Holstein bulls. ...
... In addition, most protocols for epididymal sperm freezing were developed for Bos indicus or Holstein bulls. [9][10][11][12][13][14] Cryopreservation of epididymal sperm from beef bulls of Bos taurus breeds has faced disappointingly low success, with postthaw total motility ranging from 10 to 16%. 15,16 Thus, there is a critical need to improve cryopreservation protocols for these breeds to improve postthaw semen quality, and use of a different type of semen extender may improve postthaw semen quality. ...
... 8 These differences may influence sperm response to cryopreservation and their interaction with semen extenders. Bovine epididymal sperm has been routinely cryopreserved using egg yolkbased extenders, [9][10][11][12][13][14][15][16][17] but studies testing liposome-based extenders for cryopreservation of epididymal sperm are lacking. In addition, most protocols for epididymal sperm freezing were developed for Bos indicus or Holstein bulls. ...
... In addition, most protocols for epididymal sperm freezing were developed for Bos indicus or Holstein bulls. [9][10][11][12][13][14] Cryopreservation of epididymal sperm from beef bulls of Bos taurus breeds has faced disappointingly low success, with postthaw total motility ranging from 10 to 16%. 15,16 Thus, there is a critical need to improve cryopreservation protocols for these breeds to improve postthaw semen quality, and use of a different type of semen extender may improve postthaw semen quality. ...
Article
Egg yolk-based semen extenders are commonly used to freeze ejaculated bull semen. However, liposome-based extenders have gained popularity as an alternative to animal-based products. It remains to be determined if liposome- and egg yolk-based extenders are comparable to freeze epididymal sperm (EP). We hypothesized that there are no differences in response to cryopreservation between electroejaculated (EE) and EP sperm, but that the egg yolk-based extender is superior to a liposome-based extender due its cryoprotection mechanisms. This study aimed to evaluate functional sperm parameters associated with cryotolerance in EP and EE sperm frozen with two semen extenders. One ejaculate was collected from 10 bulls by electroejaculation. Each ejaculate was divided into two aliquots, and cryopreserved with a liposome-based extender (Optixcell; EEO) or an egg yolk-based extender (Botubov; EEB). Three days later, the bulls were castrated. Sperm from one cauda epididymis from each bull were cryopreserved with Optixcell (EPO), while the other one was cryopreserved with Botubov (EPB). Concentration, motility, and morphology were evaluated before cryopreservation. After thawing, total (TM) and progressive (PM) sperm motilities, capacitation, acrosomal and plasma membrane integrity, and mitochondrial potential were evaluated and compared among treatments using ANOVA. The percentage of normal sperm did not differ between EE (76±4.7%) and EP samples (75.1±6.1%). There were fewer distal droplets in EE (11.6±3.6%) than EP (41.4±6.7%) samples (P=0.001). Post-thaw TM (P=0.015), but not PM, was higher in EPB (58.6±5%) than EPO (41.8±4%) sperm cryopreserved in Botubov than Optixcell. The percentage of sperm with intact plasma and acrosomal membranes was higher with Botubov (in EEB 60±3%, EPB 59.7±4%) than Optixcell (EEO 37.8±6%, EPO 25.8±6%) and EP sperm frozen in Botubov than Optixcell (P=0.001). There was no difference in motility and acrosomal integrity between ejaculated and epididymal sperm. The percentage of membrane-intact capacitated cells also differed with treatment (EEB 2±0.4%, EEO 6.9±2.4%, EPB 0.9±0.3%, EPO 4.2±1.4%; P=0.009). EP sperm had fewer cells with high mitochondrial potential (EPB 6.6±4.4%, EPO 14.6±6.2%) than EE sperm (EEB 46.7±9.8%, EEO 41±14.6%), with no difference between extenders (P=0.026). In summary, samples cryopreserved in the egg yolk-based extender had higher total motility, less cryocapacitated sperm, and a greater number of intact acrosomes. The egg yolk-based extender was more effective at mitigating the effects of cryocapacitation on membrane fluidity and acrosomal integrity. Ejaculated sperm suffered less damage to their mitochondria than epididymal sperm.
... The recovery, preservation, and use of epididymal sperm are essential tools to preserve genetic stocks of valuable domestic or wild animals [1][2][3] under adverse conditions [4] and also as an alternative source of gametes in cases of human infertility [5,6]. Previous studies have already demonstrated the viability of bovine spermatozoa collected directly from the tail of epididymis [7,8], but in most cases, the gametes were obtained immediately after slaughter or castration, or from epididymides that had been refrigerated at 5 C for long periods [9,10]. Few studies [11,12] have reproduced the real and more frequent situation, of the need for gamete utilization, i.e., accident, death, or inability to obtain spermatozoa in the conventional way, when structures are exposed to ambient temperature before preservation. ...
... The spermatozoa retrieved from the tail of epididymis have special features, such as the absence of seminal plasma and large numbers of distal cytoplasmic droplets, which necessitate special handling, both for cryopreservation and IVF [9,13]. Although it is a relatively new practice, good results have been achieved in cryopreservation of bovine epididymal spermatozoa using TRIS-based diluents containing egg yolk, glycerol, and citric acid [1,8,10,14]. ...
... The degree of cryopreservation damage also depends on cooling-thawing rates and freezing medium composition determining their protective properties [9,10]. Cryoprotectant media for many animal species, including dogs, most often include glycerol and egg yolk [11][12][13]. Egg yolk amino acids are widely known to have cryoprotectant and antioxidant effects on dog spermatozoa, however, it is difficult to standardize by compounds composition, which varies with bird diets [14]. Moreover, the use of chicken yolk always carries the risk of cross-contamination of samples with various infectious agents and has possibility to change spermatozoa membrane properties while acting with foreign egg yolk proteins [15]. ...
Article
Full-text available
Egg yolk is a very common supplement of extenders aimed to protect sperm from cryoinjury, but due to their biological risks and difficulties with media standardization, there is a search for alternative. In addition, sperm cryoresistance can be affected by the initial decrease of their functional characteristics caused by age. The aim of this work was to evaluate the efficiency of using dextran (molecular weight 500 kDa) in the extenders instead of egg yolk for the cryopreservation of spermatozoa of dogs (Chinese Crested breed) of different ages. The obtained ejaculates were divided into three groups depending on the animal’s age: 1–3, 4–6 and 7–10 years old. Sperm was cryopreserved by using 7% glycerol and 20% egg yolk, or 20% dextran. The cryoresistance of spermatozoa of the oldest age category was dramatically decreased, which was manifested in their morphology, motility, and DNA fragmentation rate. There were no differences between the cryoprotectant effect of the dextran-based extender on spermatozoa and the egg yolk-based extender in all age categories of dogs. However, given the benefits of dextran-containing media, its use for the cryopreservation of canine spermatozoa has potential benefits that need to be confirmed by sperm fertilization outcomes.
... Spermatozoa present in the cauda epididymis can be recovered and used at post-mortem when the ejaculated sperm are not available such as in the case of the sudden death of genetically invaluable livestock males 1 and in endangered wild species. 2 In this regard, establishing efficient, simple and inexpensive freezing methods can lead to a more widespread application of epididymal spermatozoa cryopreservation and has a great potential. ...
Article
Background : Establishing an efficient, simple and inexpensive method for freezing ram epididymal sperm so that the quality and fertility of sperm could be maintained for a longer period after thawing is of great practical value. Objectives : To optimize freezing and thawing protocol for ram epididymal sperm using either ethylene glycol (EG) or glycerol (GLY) as cryoprotectants (CPAs). Then, to evaluate the post-thaw longevity and in vitro fertility of sperm that were frozen and thawed according to the optimized protocol. Materials and methods : At first, an optimum protocol for freezing and thawing sperm using EG or GLY were investigated, and the next experiments were performed using the sperm that had been frozen and thawed according to the optimized protocol for each CPA. In the next experiments, frozen-thawed and fresh sperm were diluted in an isotonic culture medium and subsequently incubated at 39°C for 4 hours. The motility characteristics and functional membrane integrity (FMI) of sperm were evaluated after thawing, after dilution (t0), and after incubation (t4). The in vitro fertility of the sperm was assessed at t0 and t4. Results : For both CPAs, the highest motility parameters and FMI was found for sperm frozen at 3 cm above LN2 and thawed at 50 and 65°C (P<0.05). In comparison to the sperm of GLY group, sperm of EG group had higher total and progressive motility at t0, as well as higher FMI, total and progressive motility, and linearity at t4 (P<0.05). Fertility of frozen-thawed sperm was higher than that of fresh sperm at t0 (P<0.05). Incubation treatment increased the fertility of fresh sperm while decreased the fertility of frozen-thawed sperm, and this decline was more severe in GLY than in EG group. Conclusion : Based on the findings, EG can be a more suitable CPA for freezing ram epididymal sperm. This article is protected by copyright. All rights reserved
... In addition, egg yolk also contains glucose which is preferentially used by bovine spermatozoa over fructose which is naturally found in semen (Toelihere, 1985;Thun et al. 2002;Lopes et al. 2015). The drastic decrease in spermatozoa motility observed during the 4 th and 5 th day with An-dromed® diluent may be attributed to the presence of glycerol as an ingredient. ...
Article
Full-text available
Bali bull has great potential to be developed as a national meat source in Indonesia which can address concerns of meat importation. Bali bull farming only requires small holding system, has high fertility and low calf mortality as its advantages and is a growing enterprise by way of artificial insemination (AI) with semen collected from phenotypically superior bull sires. However, efficient means of preserving Bali bull semen quality using diluents still requires exploration at a national level. The purpose of this study was to determine which of the selected diluents will best preserve the quality of local Bali bull semen over a period of five days at 4 ˚C. Andromed® and egg yolk-tris diluents were compared in preserving quality of semen collected from seven-year old bulls with 70% minimum motility. Spermatozoa motility, viability, and abnormality were observed and compared. This research used factorial complete randomized design with two factors in which each factor has 2 levels and 5 levels with three-times treatment. Results of the study showed Bali bull spermatozoa demonstrated 51.66% motility, 59.13% viability, and only 22.68% abnormality until day five in egg yolk-tris diluent. In comparison, Andromed® diluent demonstrated 23.33% motility, 45.59% viability, and 23.15% abnormality under similar conditions. To conclude, egg yolk-tris is a superior diluent when compared to Andromed® in preserving local Bali bull semen quality by better maintaining spermatozoa motility, viability and lower percentages of abnormalities when stored for five days at 4 ˚C. This data provides useful information for bull farmers, in particular Bali bull farmers in Indonesia in the practice of AI to consider carefully the choice of diluent relative to storage time and temperature in improving cattle farming practices for increased meat productivity in the region.
... Preserving epididymal spermatozoa has an application whenever the ejaculated sperm is not available such as post-mortem recovery or sudden death of genetically invaluable male in farm animals (Ehling et al., 2006) and in endangered wild species (Lopes et al., 2015). In this regard, establishing efficient methods to preserve the fertility of epididymal spermatozoa has a great applied potential. ...
Article
Post-mortem recovery and freezing of epididymal spermatozoa is an option (in some instances the only option available) for preserving genetic material in wild species at risk of extinction and in farm animals when a genetically valuable male dies accidentally. Evidences exist that oxidative stress may involve in the cell death and reduced functionality of spermatozoa following cryopreservation. This study was aimed to improve the freezability and post-thaw fertility of ram epididymal spermatozoa by the addition of antioxidants into the freezing extender. Ram epididymal spermatozoa were frozen either at the presence of L-glutamine (2.5 or 5 mM), sericin (0.25 or 0.5%), reduced glutathione (GSH, 2.5 or 5 mM), or in freezing medium without supplement (Control). Immediately after thawing, the motility, functional membrane integrity and DNA damage of the thawed spermatozoa were not significantly different between groups. After 3 h post-thaw incubation at 39 °C, however, the total motility, progressive motility, and some kinematic parameters of GSH5 group were higher than other groups (P < 0.05). Oocytes inseminated with frozen-thawed spermatozoa of GSH5 and Control group had lower cleavage and blastocyst (blastocyst/oocyte ratio) rates compared to those inseminated with fresh epididymal sperm (P < 0.05). However, the blastocyst rate (both blastocyst/oocyte and blastocyst/cleavage) in GSH5 group was higher than control (P < 0.05). In conclusion, the addition of GSH to the freezing extender could improve the in vitro fertility of frozen/thawed ram epididymal spermatozoa.
... Several researches have been done to compare the type of semen extenders used for epididymal spermatozoa collection. Lopes et al., (2015) and Krishnakumar et al., (2011) Seven testes were used in both flushing and mincing groups. Five replicates of sperm motility and motility parameters in each bull were evaluated. ...
Article
In this study, two epididymal spermatozoa recovery methods in relation to sperm number, motility, viability and acrosome reaction were examined. Seven bulls were castrated and 7 testicles with epididymides were transferred to the laboratoy. Epididymis in each bull was randomly used for flushing and mincing methods with semen extender (Optixcell, IMV, France). The recovered spermatozoa with adjusted sperm concentration to 40 × 106 cells/mL was diluted with optixcell and cryopreserved. In experiment 1, the difference in the total number of spermatozoa using flushing and mincing methods was insignificant (2570.0 and 2505.2 × 106 cells/mL, respectively). For experiment 2, the percentage of motile spermatozoa and motility parameters between flushing and mincing methods were studied through the use of sperm class analyzer after frozen-thawing. The percentage of total motile sperm between flushing and mincing methods was almost the same with 89.5±12.8 and 91.4±7.9%, respectively. The same is the case with experiment 3 wherein the viability and acrosomal integrity of frozen-thawed epididymal spermatozoa by flushing and mincing was insignificantly different. The results from the study showed that both flushing and mincing methods can be used for epididymal spermatozoa recovery in bull.
... Usually, bullrings are far from ART facilities and thus, transport and cooled storage of the epididymis become necessary to allow for sperm harvesting (Malcotti et al., 2012). Fortunately, epididymal sperm can be successfully cryopreserved and used for artificial insemination or in vitro production of embryos (Martins et al., 2007;Lopes et al., 2015), although the maximum refrigerated epididymal storage time still needs to be determined in the Lidia bovine breed. ...
... Usually, bullrings are far from ART facilities and thus, transport and cooled storage of the epididymis become necessary to allow for sperm harvesting (Malcotti et al., 2012). Fortunately, epididymal sperm can be successfully cryopreserved and used for artificial insemination or in vitro production of embryos (Martins et al., 2007;Lopes et al., 2015), although the maximum refrigerated epididymal storage time still needs to be determined in the Lidia bovine breed. ...
Article
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The Lidia bovine breed is an important hallmark of the Spanish cattle industry. Bulls are selected based upon aggressiveness and epididymal sperm cryopreservation is the way to obtain and store their genetics. There are not specifically designed protocols yet to perform Lidia bull sperm cryopreservation. The present study aimed to determine if a tris-fructose-citrate-egg yolk (20% v/v; TFY) extender supplemented with 7% glycerol (TFY1) or 3.5% glycerol plus 3.5% dimethylformamide (DMF; TFY2) are suitable media for cryopreservation of epididymal Lidia bull sperm. Moreover, the effect of N-acetylcysteine (NAC), a potent antioxidant, was evaluated. The epididymis were stored at 4°C for 24, 48, 72 or 96 h, and both freezing media were tested as such or supplemented with 1 or 2.5 mM of NAC. Our data demonstrated that post-thaw viability was well maintained (TFY1: 50.8% ± 1.9 at 24 h and 52.4% ± 0.8 at 96 h and TFY2: 52.6% ± 1.6 at 24 h and 56.1% ± 1.8 at 96 h; mean % ± SEM; p>0.05) as also were total and progressive sperm motility, high mitochondrial membrane potential, ROS production, DNA status and acrosomal intactness of Lidia bull sperm up to 96 h of epididymal storage, all extender variations being similar (p>0.05). In conclusion, the use of TFY medium supplemented either with 7% glycerol alone or the combination of 3.5% glycerol and 3.5% DMF were equally safe choices for epididymal Lidia bull sperm cryopreservation, and NAC addition did not significantly improve sperm post-thaw quality.
... In sheep, Kaabi et al. (2003) observed samples storage at 5°C had higher fertilizing capacity compared with samples stored at room temperature. Lopes, Soares, Ferreira, and Rocha (2015) showed results of total blastocysts similar to our study, with bovine epididymal samples stored at 5°C until the cryopreservation process; however, sperm evaluation was performed only with conventional tests, differently from the present study. These results show that low temperatures during the storage are important to improve the fertility potential of cryopreserved samples from epididymal spermatozoa in bovines. ...
Article
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.
Article
This work aimed to evaluate the effect of stabilisation times, glycerol concentration, and the catalase and superoxide dismutase supplementation of diluent on parameters of frozen-thawed spermatozoa from epididymis of Nelore bulls: Experiment 1: spermatozoa diluted in Tris-egg yolk with glycerol (3%, 5% or 7%) and stabilisation times (0, 2 or 4 hr at 5°C); Experiment 2: Tris-egg yolk only, Tris-egg yolk with catalase (CAT, 50 or 100 U ml(-1) ) or superoxide dismutase (SOD, 50 or 100 U ml(-1) ). Frozen-thawed spermatozoa were evaluated for kinetic parameters, plasma membrane and acrosome integrity, mitochondrial activity and IVF capacity. ALH and BCF were affected (p < .05) by glycerol at 3% after 4-hr equilibration time and 7% after 2-hr equilibration time. Glycerol 3% had lower (p < .05) iPM and iAc after 4 hr. Glycerol 5% had greater (p < .05) hPMM after 4 hr and iAc after 2 hr than at 0 hr. SOD 100 U ml(-1) had lower (p < .05) linearity and wobble compared to control group. No was observed differences to fertilisation rate (p < .05) among groups. In conclusion, glycerol 5% in Tris-egg yolk extender for 4 hr is suitable for the preservation of sperm kinetics and membrane integrity. CAT (50 and 100 U ml(-1) ) or SOD (50-100 U ml(-1) ) had no beneficial effects on sperm kinetics, plasma and acrosomal membrane integrity, mitochondrial activity or the capacity for IVF of frozen-thawed spermatozoa from epididymis of Nelore bulls.
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Heat stress is quite tough on animal productivity. The exposure of animals to hyperthermia or hypothermia is associated with morphological and physiological modifications. This experiment aimed to study the HSPA1A and HSP90AA1 gene expressions of Mertolenga cows after heat exposure. The animals were subjected to the heat tolerance test, where rectal temperature and respiratory rate were measured, and blood samples were collected. In all samples was carried out the erythrocytes lysis to obtain the buffy-coat. The RNA was isolated by the TRIzol method and RT-PCR performed with SuperScript III after digestion with DNase I. The qPCR apparatus took place in 7500 Real Fast Time, using TaqMan Gene Expression Assays for HSPA1A and HSP90AA1 target genes, ACTB and PPIA as endogenous genes. The ΔCt (Cttarget - Ctendogenous) were calculated as well as gene expression through the 2-ΔΔCt method. Statistical analysis was performed using linear mixed models. There were significant differences between treatments for respiratory rate, which with the absence of significant differences for rectal temperature, allows us to state that the homeotermia was achieved by tachypnea. mRNA-HSPA1A and mRNA-HSP90AA1 values were higher in T ≥ 46 °C, as expected. These data are evidence of high thermoregulatory capacity of the Mertolenga breed.
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Assisted reproductive techniques (gamete cryopreservation, artificial insemination, embryo transfer, and in vitro fertilization) allow to propagate small fragmented populations of wild endangered species or domestic breeds. There are the best way for producing several offspring from selected genitors in order to avoid inbreeding depression. However, few mammalian species have been well studied for their reproductive biology whereas huge differences have been observed between these species. Furthermore, materials, methods and experimental designs have to be adapted for each case and each limiting factor (wildness, poor quantity of biological material, disparate locations). Genome resource banking is currently arising and the most applied reproductive biotechnology remains artificial insemination. Assisted reproductive techniques currently developed in domestic species (intracytoplasmic sperm injection, nuclear transfer) may offer new opportunities for the propagation of endangered species.
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The development of a reliable technique to freeze epididymal semen would provide a unique opportunity to preserve valuable genetic material from unexpectedly lost stallions. The aim of this study was to compare the apoptotic indices of sperm obtained from ejaculate, sperm recently recovered from the epididymides (EP), and sperm recovered from epididymides stored at 5°C for 24 hours (EP-stored). For the first category, two ejaculates from seven stallions were collected and then submitted to cryopreservation using an egg yolk-based extender. One week after the last semen collection, the stallions were submitted to bilateral orchiectomy, and sperm from one of the cauda epididymis was harvested immediately after castration (EP). The remaining testicle was stored in a passive refrigeration container at 5°C for 24 hours before the cauda epididymal sperm was harvested (EP-stored). Sperm harvesting from the epididymis for EP and EP-stored was performed by retrograde flushing of the caudal portion of the epididymis using a skim milk-based extender. The recovered sperm was then cryopreserved using the egg yolk-based extender. Sperm motility parameters were studied by computer-assisted semen analysis, and apoptosis was estimated by measuring caspase activity and membrane phospholipid translocation using epifluorescence microscopy. The samples were evaluated immediately (0 hour) and 8 hours after thawing. At 0 hour, no differences in sperm parameters were observed among the groups, but after 8 hours, significant statistical differences were observed in sperm motility parameters and plasma membrane integrity among the treatment groups. In addition, viable cells with no apoptotic signs were more prevalent in EP and EP-stored, suggesting that epididymal sperm is less sensitive to the cold shock caused by sperm cryopreservation.
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Cryopreservation of epididymal spermatozoa is a useful tool to preserve genetic material of valuable stallions after emergency castration or unexpected death. For that, testicles and epididymides are generally sent refrigerated to the laboratory. Collection of epididymal spermatozoa is a simple procedure that reduces the volume of the material to be shipped, and may improve the quality of the chilled epididymal sperm cells. In the present study we compared the characteristics of frozen/thawed epididymal spermatozoa after refrigeration of the epididymis or after direct refrigeration of the extended epididymal sperm cells. Ejaculated sperm samples were obtained from 10 healthy stallions with at least 15 days of sexual rest, before routine orchiectomies. Spermatozoa were recovered from the epididymal tail immediately after castration (EPI), after refrigeration of the epididymis for 24h at 4°C (EPI R) and recovered from epididymal tail immediately after castration and stored for 24h at 4°C (EPI RR). Total motility, straight-line velocity, percentage of rapid cells, viability and morphological defects were similar (p>0.05) among different treatments, and post-thaw viability was higher (p<0.05) in EPI than in the ejaculated sperm. The similarity of post-thaw parameters led us to conclude that immediate collection and refrigeration of the epididymal sperm cells or refrigeration of the whole epididymis are equally efficient as a means of transporting material for 24h before cryopreservation of epididymal spermatozoa.
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Techniques for canine semen freezing need to be optimized in order to maximize pregnancy rates and to increase the number of AI-doses obtainable from a single ejaculate. To improve these techniques, simultaneous investigation of different aspects of the cryopreservation process must be performed, to find the best combination of extender and freezing and thawing rate. Four ejaculates were obtained from each of five dogs, and split-samples were diluted in a Tris citrate glucose extender containing 0.5% Equex STM Paste and either 3 or 5% glycerol. Two ejaculates from each dog were frozen at a slow freezing rate (10 degrees C/min in the range -6 degrees to -40 degrees C) and two at a fast freezing rate (50 degrees C/min in the range -6 degrees to -40 degrees C), in 0.5 mi straws using a programmable freezer. Prior to evaluation, the straws from each freezing rate and glycerol concentration were thawed in a water-bath, either at 38 degrees C for 1 min or at 70 degrees C for 8 s. Samples of cryopreserved semen were evaluated for motility and for the proportion of spermatozoa having an intact plasma membrane, immediately after thawing and during 5 h of incubation at 38 degrees C. The higher glycerol concentration and the faster thawing rate tested favoured the recovery of living and motile spermatozoa and their survival during incubation, while the tested freezing rates had no influence on sperm post-thaw longevity.
Article
Post-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 72h, 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 x 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.
Article
In the Canadian Animal Genetic Resource Program, bull semen is donated in frozen or fresh (diluted) states. This study was designed to assess the cryopreservation of diluted bull semen shipped at 4°C overnight, and to determine the post-thaw quality of shipped semen using different straw volumes and freezing rates. Semen was collected from four breeding bulls (three ejaculates per bull). Semen was diluted in Tris-citric acid-egg yolk-glycerol (TEYG) extender, cooled to 4°C and frozen as per routine (control semen). After cooling to 4°C, a part of semen was removed and shipped overnight to the research laboratory via express courier (shipped semen). Semen was packaged in 0.25 or 0.5 ml straws and frozen in a programmable freezer using three freezing rates, i.e., -10, -25 or -40°C/min. Control semen was also shipped to the research laboratory. Post-thaw sperm motility characteristics were assessed using CASA, and post-thaw sperm plasma membrane, mitochondrial membrane potential and normal acrosomes were assessed using flow cytometry. Post-thaw sperm quality was greater in shipped semen as compared to control (P<0.001). The shipped semen packaged in 0.25 ml straws had better post-thaw sperm quality than in 0.5 ml straws (P<0.001). Freezing rate had no effect on post-thaw sperm quality. In conclusion, bull semen can be shipped overnight for subsequent cryopreservation and gene banking. Overnight shipping of semen was found advantageous for bull semen cryopreservation. Semen packaging in 0.25 ml straws yielded better post-thaw quality than 0.5 ml straws.
Article
The 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 72h 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 24h post-mortem, was higher compared to the Ex-EPID samples, although differences decreased at 48 and 72h. 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 72h. At 24h, 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 24h, 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 48h. The D320 extender preserve motility of epididymal spermatozoa but does not protect the status of the acrosome.
Article
The objective of this study was to develop a protocol for ram epididymal sperm preservation that could be applied to wild ruminants for collection and preservation of spermatozoa from dead or hunted animals. Ram testicles collected from abattoirs were used to study the effect of two transportation temperatures viz. ambient temperature (AT) and refrigeration temperature (RT) on the cauda epididymal sperm quality at recovery and during preservation up to 72 h at 4 °C. For AT the testicles were transported in normal saline in a container (17.9–21.5 °C) where as for RT the testicles were transported in an ice-chest (4.9–6 °C). The results of the current study revealed that intact acrosome was significantly higher (P < 0.01) and other quality parameters like sperm motility, live sperm count, sperm concentration and major sperm abnormalities were also higher (P > 0.05) for RT than AT. The mean percent sperm motility for RT and AT was 81.67% and 78.33%, respectively. The corresponding figures were 92.08% and 90.46% for mean live sperm, 98.33% and 90.50% for intact acrosome, 0.50% and 0.33% for major sperm defects. The percent minor abnormality was 79.50% for RT and 77.67% for AT. The most prevalent minor defect was distal cytoplasmic droplet (70–80%).
Article
The objetive of the present study was to evaluate the effect of the interval between animal's death and sperm recovery on the freezability and fertilizing ability of spermatozoa from bull epididymides stored for different periods of time. Testis from 25 bulls were collected at the abattoir 2h after the slaughter. In the laboratory spermatozoa from one epididymis were recovered and analysed for motility. The remaining epididymis was stored for 24h (G24), 48h (G48) and 72h (G72) at 5 degrees C. At the end of each time period, spermatozoa were recuperated and cryopreserved in Tris-egg yolk and glycerol. Pre-freeze and post-thaw sperm samples were taken to assess total and progressive motility, concentration, membrane integrity and acrosome integrity. For evaluation of fertilizing ability, in each time period five straws of each bull were thawed, pooled and used for in vitro embryo production. The results showed that after 48h of storage there was a decline in total motility, which did not change until 72h. Progressive motility, plasma membrane and acrosome integrity were not affected by any of the storage periods. Conversely, all sperm parameters, except progressive motility, were reduced after cryopreservation. Embryo production was less (P<0.05) in the treatments than in the reference group. However, there was no differences (P>0.05) in blastoycst rate among experimental groups. Considering all the embryos produced by epididymal spermatozoa a greater proportion of female embryos was observed, which was similar to the reference embryos. The shift observed on sex ratio toward female for those two groups was also observed when they were compared with the expected 1:1 ratio (P<0.05). The results showed the possibility to produced in vitro embryos using cryopreseved spermatozoa from epididymides and stored for long period of time at 5 degrees C. These procedures became an important tool for animal preservation when the sperm cells cannot be cryopreserved immediately after the animal's death.
Article
Bovine follicular oocytes matured in vitro were fertilized in vitro using epididymal spermatozoa from five different bulls and then cultured to the blastocyst stage in vitro. The fertilization rate, based on one pair of pronuclei and presence of one sperm tail, ranged from 55.2 to 64.3%. Embryo development (cleavage to blastocyst stage) ranged from 21.4 to 31.0% of the cultured ova reaching 8 cells at 3 to 4 d after insemination to 1.3 to 3.7% reaching hatched blastocysts at 9 to 10 d. It is concluded that individual variation among bulls is not a significant factor in fertilization and development rates of bovine follicular oocytes when epididymal spermatozoa are used.
Article
Rapidly cooling (cold shocking) washed cauda boar sperm irreversibly reduced motility and respiration and greatly increased the uptake of 45Ca2+; the plasma membranes were removed and the acrosomes detached from nuclei. The motility, respiration, and calcium uptake of the less mature caput sperm were largely unaffected; and there was little damage to the ultrastructure. This indicates that boar sperm becomes less resistant to cold shock as they mature in the epididymis. The oxygen uptake, glucose breakdown, and lactic acid production of control caput sperm was less than that of cauda sperm. This suggest that the maturation of sperm in the epididymis of the boar involves an increase in both the glycolytic and oxidative phases of glucose metabolism. The presence of 2.0 mg/ml phosphatidylcholine (lecithin) in the medium prevented ultrastructural damage to cauda sperm on cold shock, and motility and respiration were maintained at levels similar to those of control sperm. Although the presence of phospholipid reduced the large calcium influx following cold shock, it was still greater that that of control sperm. The “protective” effect against cold shock was not maintained after rewashing the sperm free of phosphatidylcholine prior to cold shock, indicating a fairly “loose” interaction of the phospholipid with boar sperm membranes that was easily disrupted.
Article
Spermatozoa recovered from the caput epididymides of four bulls were immotile, whereas sperm from the cauda epididymides averaged 41% progressive motility. The percentage of unstained or morphologically normal spermatozoa did not differ between the caput and cauda epididymides. When caudal sperm were used to inseminate 100 cows, 69% did not return for reinsemination in 60 to 90 days.
Article
The influence of prolonged storage of boar epididymides on post-thaw sperm motility, and in vitro fertilization was evaluated. Twenty pairs of epididymides were obtained from Large White boars, and spermatozoa from one of each of the pairs were immediately collected and frozen (control group). The remaining epididymides were cooled to 4 degrees C and stored for 1, 2 or 3 d, after which spermatozoa were collected and frozen (experimental groups Day 1, 2 and 3, respectively). Sperm motility was maintained throughout the dilution procedure and then dropped (P < 0.01) after freezing and thawing. During storage the motility of nonfrozen spermatozoa decreased significantly (P < 0.01), reaching a value equal to that of frozen-thawed spermatozoa on Day 3. In vitro fertilization experiments revealed significantly (P < 0.05) lower penetration rates using Day 1, 2 and 3 stored spermatozoa (12, 13 and 2%, respectively) than that of the control group (40%). Oocyte penetration ability seemed to be reflected by acrosome integrity. However, the motility of spermatozoa with the ability to penetrate oocytes in Day 1 and Day 2 groups did not differ from that of the controls. The motility of spermatozoa lacking penetration ability, on the other hand, gradually decreased as the storage period was prolonged. This suggests that the sperm motility and penetration ability are affected by different mechanisms during the cold storage of epididymides. Finally, control and experimental groups exhibited high incidences of monospermic penetration (64 to 90%) and of male pronuclear formation (67 to 71%). These data suggest that cryopreservation of spermatozoa from boar epididymides stored at 4 degrees C for 1 to 2 d can be used for conserving male germ cells when epididymal spermatozoa can not be collected immediately and cryopreserved.
Article
Among the many mammalian species that are threatened as the result of habitat destruction are numerous species of rare or little-known native livestock that possess features that render them ideally adapted to their environment. Because of the vital and valuable role many of these species play both to the ecology and economy of their native countries, attention is being directed towards initiating breeding programs that might insure their continued survival. This review introduces and highlights the importance of some of these indigenous species and outlines efforts currently underway to apply assisted reproductive technologies to their conservation.
Article
Cryopreservation imposes irreversible damage to sperm membranes, such as swelling and disruption of plasma and acrosome membranes, changes in membrane fluidity, altered influx of calcium, and changes in enzyme activity. Morphological integrity of the sperm plasma membrane has been widely studied using different techniques, including exposure of spermatozoa to hypoosmotic solutions (provides information concerning the biochemical activity of the sperm tail membrane), supravital test using eosin stain (yields information regarding sperm head membrane integrity), and Trypan-blue Giemsa stain (TBG; reveals both sperm plasma membrane and acrosome integrity). The objective of this study was to combine these tests in order to provide information about the integrity of the whole sperm surface, as well as acrosome status, and determine if the results of these tests were associated with sperm in vitro fertilizing ability. Stepwise regression analyses yielded a model in which fertility (maintain variable) was expressed as a combination of the results of different spermatological parameters (independent variables). The results of a test combining supravital eosin staining of samples previously submitted to hypoosmotic swelling test (STHOS) accounted for the greatest proportion of variation in fertilization rates (78%). Inclusion of the results of dual staining with TBG increased the proportion of variation in fertility rate that could be accounted for to 82%. Therefore, sperm plasma membrane integrity and function, and acrosome integrity can be considered important variables for normal sperm function and STHOST and TBG could be used for the prognosis of the potential fertility of bovine semen samples used for IVF or AI.
Article
The aim of this study was to assess the influence of prolonged cold storage of Iberian red deer epididymides on post-thaw sperm characteristics. Thirty-seven pairs of testes, with attached epididymides, were collected during November and December. Spermatozoa from one of each of the pairs were immediately recovered, evaluated and frozen (control group). The remaining epididymides were cooled to 5 degrees C and stored for 12, 24, 48, 72 and 96 h (experimental groups), after which spermatozoa were collected and frozen as in the control group. After thawing, sperm motility, membrane and acrosome integrities, mitochondrial function and DNA damage were evaluated. The motility of spermatozoa stored in the epididymis for up to 96 h did not decrease significantly (P>0.05) but, after cryopreservation, a decline in sperm motility was seen in spermatozoa stored for 48 h, or later. A slower decrease in sperm membrane and acrosome integrities after cryopreservation were seen as storage time progressed. Some differences were seen when different methods were used to assess the same sperm parameter although changes followed similar patterns. This was the case for acrosome integrity (phase contrast microscopy versus fluorescent lectin) or membrane integrity (hypo-osmotic swelling test or nigrosin-eosin stain versus propidium iodide). We conclude that frozen-thawed spermatozoa of Iberian red deer recovered from epididymides stored at 5 degrees C have a good sperm quality (including motility) during less than 48 h of storage for most of the sperm parameters assessed.
Article
An experiment was conducted to investigate the freezing ability of canine epididymal spermatozoa after cool storage at 5 degrees C for 2 or 4 days. Spermatozoa were collected from the caudae epididymidis from 16 dogs. Total motility, plasma membrane integrity and acrosome integrity were evaluated immediately on harvesting, and after 2 and 4 days of storage at 5 degrees C, and at 0 and 2 h post-thaw at 37 degrees C. Sperm motility decreased significantly during cold storage, compared to freshly harvested spermatozoa (P < 0.001). Although there was no significant effect of pre-freeze storage time on post-thaw motility, there was a tendency towards decreased motility in spermatozoa that had been stored for 4 days, compared to spermatozoa that were frozen immediately after collection (P = 0.09). The number of post-thaw spermatozoa with an intact plasma membrane was decreased in spermatozoa cold-stored for 4 days (P < 0.001). There was no significant effect of pre-freeze storage time on the acrosomal status of post-thaw spermatozoa. In conclusion, canine epididymal spermatozoa were stored at 5 degrees C for up to 4 days without a clear detrimental effect on post-thaw motility and acrosome integrity, but storage may have decreased post-thaw motility. Results were, however, generally low.
Article
Because of risks of disease transmission, it is not possible to move African buffalo (Syncerus caffer) within South Africa. Therefore, new ways must be found to enable exchange of genetic material and to increase genetic diversity. In this study epididymal sperm from 11 African buffaloes was exposed to 8 different pre-freezing equilibration times, using 2 different semen extenders. To test the influence of equilibration time and to find a practical way of freezing sperm in the field equilibration times between 2 and 9 h were compared. The extenders used were Triladyl and the totally defined extender AndroMed (both Minitüb, Tiefenbach, Germany). Post-thaw motility, longevity and acrosomal integrity were compared. Different equilibration times did not result in different post-thaw qualities. The use of Triladyl resulted almost always in higher post-thaw motilities and in better acrosomal integrity. Individual bulls had a significant influence on measured parameters. Results indicate that sperm flushed in the field can be stored in freezing medium for up to 9 h before being further processed and that Triladyl is superior to AndroMed when freezing epididymal African buffalo sperm. This knowledge is important to plan fieldwork, since working conditions are usually far from the ideal of a laboratory.
Article
This review describes the use of modern reproductive biotechnologies or assisted reproductive techniques (ART) including artificial insemination, embryo transfer/sexing, in vitro fertilization, gamete/embryo micromanipulation, semen sexing, genome resource banking, and somatic cell nuclear transfer (cloning) in conservation programs for endangered mammalian species. Such biotechnologies allow more offspring to be obtained from selected parents to ensure genetic diversity and may reduce the interval between generations. However, the application of reproductive biotechnologies for endangered free-living mammals is rarer than for endangered domestic breeds. Progress in ART for non-domestic species will continue at a slow pace due to limited resources, but also because the management and conservation of endangered species is biologically quite complex. In practice, current reproductive biotechnologies are species-specific or inefficient for many endangered animals because of insufficient knowledge on basic reproduction like estrous cycle, seasonality, structural anatomy, gamete physiology and site for semen deposition or embryo transfer of non-domestic species.
Article
The present study aimed to evaluate viability and in vitro fertilizing ability of cryopreserved epididymal spermatozoa obtained from dead animals. To collect spermatozoa, epididymides from three males (Bulls A1, A2 and A3) were collected at a local slaughterhouse. As a reference ejaculate from a bull with known in vitro fertility, was used. Sperm characteristics (motility, chromatin and acrosome integrity) were evaluated before and after cryopreservation. Then, frozen spermatozoa from all animals were used for in vitro fertilization. Cleavage and blastocyst rates at 48 h (day 2) and 168 h (day 7) post in vitro insemination, for bull A1 (82.1 and 38.6%) and A2 (80.7 and 33.8%) were similar (P>0.05) to the reference bull (88.9 and 57.2%). Bull A3 had the lesser cleavage (42.0%) and blastocyst (26.1%) rates. The results showed that epididymal spermatozoa from dead animals can be successfully cryopreserved and used in vitro production of embryos.
Article
Post-mortem spermatozoa recovery is an important technique for obtaining germplasm reserves from genetically valuable animals or endangered species. However, there are many factors that influence the outcome of this technique. We have studied the effect of the interval between animal's death and sperm recovery (0, 24 or 48 h) on the quality and freezability of ram spermatozoa from cauda epididymidis. Storage temperature of epididymis (room temperature or 5 degrees C) was also analysed. Spermatozoa were diluted with Tes-Tris-Fructose solution supplemented with egg yolk (10%) and glycerol (4%), and frozen using a programmable biofreezer (-20 degrees C/min). Pre-freeze and post-thaw sperm samples showed viable spermatozoa up to 48 h after the animal's death, although their quality declined significantly as post-mortem storage time increased. Epididymis sperm stored at 5 degrees C showed better motility and a lower percentage of abnormal forms than epididymis stored at room temperature after 24 and 48 h. The fertilizing ability of cauda epididymis ram spermatozoa obtained at 0 and 24h after the animal's death is similar to that of ejaculated spermatozoa. Therefore, a good protocol for post-mortem semen collection in rams when epididymal spermatozoa cannot be collected immediately, is to preserve the epididymis at 5 degrees C and process the samples in the first 24h after the animal's death.
Article
We have carried out a study on the influence of prolonged cold storage (5 degrees C) of Iberian red deer epididymides on post-thaw sperm motility and DNA integrity. Twenty-nine pairs of testes, with attached epididymides, were collected during November and December. Spermatozoa from one of each of the pairs were immediately recovered, evaluated and frozen (control). The remaining epididymides were cooled to 5 degrees C and stored for 24, 96 and 192 h (experimental groups), after which spermatozoa were collected and frozen. Samples were evaluated before freezing, after thawing, and after a 2-h period of incubation at 37 degrees C. Motility was evaluated by means of a CASA system and chromatin stability was assessed following the Sperm Chromatin Structure Assay (SCSA). Our results showed that, during the first 96 h, the motility (total and progressive) did not significantly decline when assessed after cryopreservation, although there was a significant decline when epididymides had been stored for 192 h at 5 degrees C (P<0.001). The present study demonstrates that motility and DNA status of thawed spermatozoa collected from refrigerated epididymes, at least 96 h post-mortem, were good enough to consider their eventual use. Most importantly, sperm DNA integrity after thawing was apparently not affected by storage time, even after 192 h.