Place of study and animals

For the present study, a total of 50 pairs of bull testicles diagnosed healthy on the ante-mortem examination, of different racial types and ages, were used in the Municipal slaughterhouse of Loja canton, while the processing of the samples was carried out in the Laboratory of Animal Reproduction, located in Punzara Experimental Farm of the National University of Loja, Ecuador.

Collection and transport of samples

The testicles were collected from 50 bulls of different races and aged between two and four years, during 10 visits to Municipal slaughterhouse of Loja. The steps followed for the collection of the testicles were: collection of scrotal sacs with their testicles included; each scrotal sac was identified with the number corresponding to the order in which they were collected, in addition to the date and time of collection; each scrotal sac was wiped with paper towels, the testes/epididymis were ligated into the spermatic cord; each was placed in sealed and identified plastic bags and then transported from the collection site to the laboratory within a maximum of 30 minutes at an initial temperature of 35 °C. To achieve this temperature was used thermal bags with tempered water at 37 ºC.

Once in the laboratory, each testicle was placed in a Petri dish, where an external wash of the epididymis tail was performed with physiological saline solution. To remove blood and external contaminants, the connective tissue that covers the tail was removed by careful dissection, avoiding the rupture of the blood vessels and the epididymal duct; the testes were then separated from the epididymides and washed with saline; a dissection of the epididymis tail was performed, removing the tunica albuginea, thus leaving a free portion of the vas deferens. Then the secretion of the same was obtained according to each method of collection studied, ie: collection of retrograde flow and slicing of the epididymis (Slicing), proceeding as follows:

First method (retrograde flow collection)

The epididymis, already separated from the testis, was placed in a Petri dish, with ringer lactate preheated at 37 ° C, for the purpose of washing blood residues; then the nearest portion of the mid-tail of the epididymis was located to make a cross-section with the scalpel, before the diameter of the epididymis was reduced. In order to obtain as much spermatozoa as possible, a needle (20 gauge, 21, 22, or 23, according to the internal diameter of each vas deferens) was placed into the lumen of the free portion of the vas deferens. A syringe was fitted with 10-15 ml of "PBS washing medium" at 37 °C with a clamp being placed against the needle on the walls of the vas deferens, thereby preventing the loss of the wash liquid by reflux. The fluid obtained with epididymal spermatozoa was centrifuged at (300g/5min) to concentrate the sample. The supernatant obtained was removed and discarded, at a concentration of 1: 1 at 37 °C.

Second method (slicing of the epididymis)

The epididymis was aseptically separated from the testis, placed in a Petri dish with lactated ringer at 37 °C, to wash away blood residues. Spermatozoa were recovered by the slicing method, with a surgical scissors in a Petri dish, containing 15 ml of PBS medium at 37 °C; this content was sucked into a 20 ml syringe. To be purified through a sterile filter; it was transferred to falcon tubes (15 ml), and then centrifuged at a rate of (300g/5min). The obtained supernatant was removed and discarded; and a pre-dilution with AndroMed in a ratio of 1: 1 at 37 ° C was performed.

Preparation of the diluent

2 ml of AndroMed was poured into a graduated falcon tube, 8 ml of doubly distilled water, tempered at 37 °C, was added to the concentrate and mixed with the aid of the vortex (mechanical stirrer) until the dilution was homogenized. This dilution was performed for each sample individually, the same one that should be prepared in a water bath between 35 and 37 ºC; then the pre-dilution of the semen was performed in a 1: 1 ratio; for this the diluent should have the same temperature as the sperm. After the semen evaluation, the final dilution was immediately carried out, which consisted of 2 ml of prediluted semen plus 8 ml of the prepared diluent. This final dilution must progressively decrease temperature until the laboratory temperature (18 to 22 °C) is reached. Subsequently the temperature is lowered with cooled water (8-10 °C), the temperature must be equilibrated for at least 1 hour, then cooled to 5 °C.

Microscopic evaluation of the seminal samples

To perform the microscopic evaluation it was necessary to wait between 40 and 45 minutes in order to observe the movements of the epididymal spermatozoa, during which time the seminal samples and the material used in a water bath at 37 ºC were maintained; after this time it was possible to estimate the percentages of motile and progressive movements of the sperm cells; 5% eosin and 10% nigrosin were used for the staining and smearing of the samples; the microscopic evaluation was done with a 40X and 100X lens. The following characteristics were analyzed: volume, concentration, morphology, vitality, mass motility and individual motility.

The result obtained from the sperm concentration was in millions/ml

Post cooling evaluation

The evaluation of individual post-refrigeration motility was performed, each group studied (2, 4, 8, 10, 24 hours). The evaluation of the seminal quality was done in fresh and every 24 hours after refrigeration for 5 days; of the sample refrigerated at 5 °C was taken, 0.5 ml of semen diluted in an eppendorf tube, which was tempered at 37 °C for 3 minutes in a water bath; the tube was then removed from the water bath, dried with a paper towel, then 10μl of semen was taken and placed in the container and tempered and placed the coverslip; this evaluation was done on the thermal plate (Chamba Ochoa, 2017).

Statistical analysis

For the analysis of data, the statistical program IBM SPSS Statistics 2.0 was used. For the description of the data were used averages and the standard deviation of each group studied, in cases where the information obtained corresponded to percentages, these were transformed according to the binomial model of parameters. All data were assessed for normality by the Shapiro-Wilk test. The variables that did not pass in the normality test (non-parametric data) were evaluated by the ANOVA on Ranks test. The variables that passed the normality test (parametric data) were evaluated by the One Way ANOVA test. When the ANOVA indicated a significant effect, the values were compared by applying Tukey's mean comparison test. Finally, the Pearson test was performed to establish the correlation between the measured variables. The level of significance was considered P <0.05 (SPSS/PC, 2012).

Morphometric characteristics of testis and epididymis

In the study of the morphometric and functional characteristics of the testes/epididymis and recovered spermatozoa, through the use of two methods of sperm retrieval of epididyms of bulls slaughtered in the Camal refrigerator of Loja; as part of the descriptive analysis, an average of 33.81 ± 5.9 cm was determined for scrotal circumference; 11.94 ± 5.8 cm for testicular length; 6.1 ± 4.4 cm for testicular width and 580 ± 50 g of testicular weight; 62.76 ± 4.2 g for epididymis weight and 17.28 ± 6.3 cm epididymis length. Data that relate to those indicated by ^{Saavedra et al., (2012}), where it indicates that the weight of each testicle is around 250 to 500 g, the testicular length between 11 and 17 cm and the width between 5 to 8 cm, likewise (^{Sudheer 2000}), obtained averages of 31.80 cm for scrotal circumference, 11.36 cm for testicular length; 5.56 cm for testicular width or depth; and 267.12 g for the weight of each of the testicles; 1.96 cm in length of the epididymis tail and 30.06 g for the weight of the epididymal tail. Also, (^{Rodríguez et al., 2000}), obtained averages for scrotal circumference of 31.25 cm, weight of epididymal tail 32 g and 16.63 cm for the length of the epididymis. The testes were evaluated within the scrotal pockets, measuring scrotal circumference, length, width and testicular weight; as well as length and weight of the epididymis.

Comparative study of the methods of seminal collection of post-mortem epididyms (0 hours)

Using the retrograde flow technique was collected, (Table 4) on average 2.0 ± 0.5 ml with a concentration/ml of 64.04 ± 1.3 x 10⁹ spermatozoa/animal, and using the slicing technique of the epididymis (Table 5). An average of 2.2 ± 0.4 ml was collected with a concentration/ml of 62.12 ± 2.8 x 109 spermatozoa/animal, corresponding to the number of cells obtained in an ejaculation (^{Gonçalves et al., 2008}). Considering that the epididymis is capable of storing several ejaculations, the quantity obtained could be higher however; the bulls used came from a slaughtering plant, where reproductive quality is not a considered index (Table 3).

It was decided to use the average/animal, since according to (^{Goovaerts et al., 2006}) one epididymis can not be control of the other, since there are significant differences in the quantity and quality of the cells collected from the epididymis of the same animal.

The fresh sperm samples presented superior results in all the parameters compared to the results of the evaluation with refrigerated semen.

Semen evaluation obtained by retrograde flushing

Microscopic evaluation of the semen obtained by crushing the epididymis (slicing)

Sperm motility is essential for the sperm to reach the uterine environment and the fertilization site, being considered the most important criterion in the evaluation of sperm cells before and after cryopreservation (^{Siqueira et al., 2007}).

In the present study microscopic characteristics presented similar percentage motile of mass motility within each group, at two post-mortem hours of 86 ± 6.1% (Table 5), for the retrograde (P1) method and 84 ± 4% (table 6), in the method of epididymis slicing (P2), similar to those obtained by ^{Chavez (2008}), who obtained 90% of mass motility in Lidia bulls, considered to be a very good descriptive value. At four hours post-mortem, 76 ± 5.7% (P1) and 72 ± 3.8% (P2) were obtained, with a good descriptive value. At 56 hours post-mortem, 56 ± 5,5% (P1) and 54 ± 3,5% (P2) were obtained, and at 10h and 24h a mean mass motility of 40 to 48% with a regular descriptive value. These descriptive values are in accordance with the reference values presented by ^{Baracaldo (2007}), who indicate very good descriptive values of 80-90%, good of 60-79%, regular of 40-59% and bad less than 40%.

Regarding the individual motility of fresh spermatozoa collected from the epididymis at two hours of storage, it was elevated, 76 ± 5.9% (table 5), with retrograde flow technique and 74 ± 5% (table 6). technique of slicing the epididymis; which can be considered within normal limits for the species, (^{Melo et al., 2008}); values slightly below the 80 % found by ^{Chavez (2008}); (P1) and 64 ± 4.3% (P2), data similar to those obtained by ^{Sánchez et al., (2010)}, who performed a study to 28 post-mortem Lydia bulls, obtained means of 60 ± 6.1% of individual motility, with a good descriptive value; in the third group at 8 hours post-mortem presented 47 ± 4.4% (P1) and 46 ± 3.9% (P2) regular; and at 10h and 24h post-mortem values were obtained of 28 and 41% respectively; giving a poor descriptive value as reported by ^{Barth et al. (2000}).

Individual motile readings that were made for both the retrograde flushing protocol and the epididymal slicing protocol did not maintain a uniform pattern; this could be related to the variability between slaughter bulls, non-breeding bulls, and even more so in this study in which we do not know the specific origin of these males destined for consumption and that we understand did not have an adequate diet, which could have affected in some way their reproductive capacity (^{Albers and Barrios 2006}).

Post-refrigeration semen quality at different evaluation times

The percentage of individual motility of the spermatozoa obtained by retrograde washing and slicing of the epididymis (slicing), after being refrigerated, the samples were evaluated every 24 h, within each method of collection (Table 6, 7 y 8).

The percentage of live spermatozoa, determined at 2h 86 ± 3% (P1) and 82.66 ± 2% (P2); (P1) and 72.2 ± 1.8% (P2), data related to those obtained by ^{Chavez M. (2008}), who in their study determined 70 and 80%; similar values found by ^{Morillo et al. (2012}), who said that the minimum acceptable percentage for live spermatozoa should reach 70%.

^{Barrios (2002}), reported a sperm concentration of 40 to 72 x 10⁹ spz/ml in the present study we determined 56.2 to 69 x 109 espz/ml, higher values than the previous ones and those found by ^{Rodríguez (2000)}, who reported 15 to 40 x 109 esp/ml. However, ^{Castro et al. (2009}) found averages of spermatozoa collected from the tail of the epididymis of 1.7 x 109 espz / ml, with a minimum of 0.26 x 109 spz / ml and a maximum of 4.2 x 109 / ml. In other studies, ^{Sánchez et al., (2010)} obtained a spermatic concentration of 380.5x109 spz/ml values lower than those obtained in the present study and that of ^{Chavez (2008}), who reported of 600 to 1800x10⁶ spz/ml.

As for morphology, the percentage of abnormal spermatozoa was 7.92 ± 1.2%, for semen obtained by retrograde flushing and 9.64 ± 1.6% for the semen obtained by the epididymis slicing, ^{Barth (2000}) and ^{Anel et al. (2002}) confirm that the total of abnormalities should not exceed 30%; suggesting that when there are few abnormalities it is sufficient to count 100 spermatozoa and when we find large numbers it is advisable to count 300 or more.

^{Castro et al., (2009}) studied the viability of bull spermatozoa, collected from epididyms refrigerated at 4 °C, 24 hours after slaughter and found results similar to that of semen ejaculated. However, in this study the best results were obtained in groups one and two, within the protocols evaluated; at 24 h, a viability was determined within protocol one (P1) 69 ± 5.8% (G1) and 55 ± 4.3% (G2); 57 ± 4.6% (G1) and 45 ± 4% (G2), in protocol 2 (P2) at 48 h; groups 3,4 and 5 did not present individual motility.

^{Martins et al. (2009}) determined that after 48 hours of refrigeration storage, there is a total decrease in motility, which is in agreement with the results obtained in this work, as confirmed by ^{Anel (2002}). , depending on the interval between sperm recovery and death, sperm motility will decrease significantly between 48 hours.

The results showed that there is no significant statistical difference between methods of sperm collection on the spermatozoa viability. Despite this, the viability results were twice the sperm mortality, indicating that the epididymis spermatozoa, after cooling, behave in the same way as the ejaculates (^{Celeghini et al., 2008}), in which immobile sperm can still be viable (^{Chavez, 2008}).