Care and well-being

The axolotl management was carried out in the Center for Biological and Aquaculture Research of Cuemanco (CIBAC-UAMX) facilities, in accordance with the Management Plan, authorized by the Ministry of Environment and Natural Resources (SEMARNAT) to the Unit of for the Management and Conservation of Wildlife (UMA) CIBAC, with registration DGVS-CR-IN 0952-DF/07/UMA CIBAC.

Accommodation and collection of spermatophores

The axolotls were housed for monitoring for one year individually in 60 L containers, at a temperature of 18 °C, with a photoperiod of 12 h light and 12 h of darkness. To stimulate spermatophore release, the males were transferred to a glass container with a capacity of 700 L of water, which was conditioned with sandy soil and aquatic plants. During darkness hours, using a 0.25 HP chiller (Ártica Resun CL-600), the water temperature was reduced; in the same way for all specimens from 18 to 14 °C, introducing 3 females per male. The spermatophores were recovered from the bottom of the container, at the beginning of the next 12 hours of light.

Spermatophore management and sperm collection

The spermatophores released by each specimen were collected at 5 °C in 2 ml of Simplified Amphibian Ringer (SAR) medium, composed of 113 mM NaCl, 1mM CaCl, 2.0 mM KCl and 3.6 mM NaHCO, with 220 mOsmol kg^-1. Spermatozoa were obtained by placing the spermatophores of each specimen in a 4-well Nunc^MR box with 0.5 ml of SAR medium; in the first well, they were washed to remove organic matter residues; In the second well, the gelatinous material (glycoproteins) was removed to obtain the cap containing the sperm; in the third well, 0.5 ml of 20% Sodium Hydroxide (NaOH) was used to soften the cap for 10 min (^{Taku et al., 2004}); in the fourth well with 0.5 ml of SAR medium, the spermatozoa were extracted by cap maceration. By aspiration, the sperm were extracted from the supernatant, then they were filtered with a 30 µm mesh and the total sperm were recovered from the spermatophores of each release in an Eppendorf tube with 500 µl of SAR medium at 5 °C, to make a pool sperm of each release.

Sperm cryopreservation

Each sperm pool, kept at 5 °C, was adjusted with 6% Dimethylacetamide (DMA), then 0.25 ml straws were filled, to maintain equilibrium for 10 min at 2 °C, then they were placed at 5 cm on steam of Nitrogen at -76 °C for 15 minutes and subsequently submerged in liquid nitrogen at -196 ° C, to be cryopreserved for 30 days until later thawing at 15 ° C for 5 min (^{Atencio et al., 2013}).

Basic sperm evaluation

The percentage of live spermatozoa was determined through a smear that was made with a 1:5 mixture of spermatozoa with eosin-nigrosin staining, in which 100 spermatozoa were counted under the microscope at 40X. Live spermatozoa were considered, those not stained and dead, those that presented staining. Sperm morphology was evaluated in the same staining, to determine percentages of sperm with morphological alterations in the head, neck or flagellum regions (^{Tanisław et al., 2017}).

Membrane carbohydrate distribution

With the use of lectins from Triticum vulgaris agglutinin (WGA), with affinity to N- Acetylglucosamine residues and Arachis hypogaea (PNA) with affinity to β-galactose, conjugated to fluorescein isothiocyanate (FICT), it was intended to determine the carbohydrate presence that have been reported are receptors for recognition between gametes (^{Herrera et al., 2017}) and as structural parts of the spermatic plasmalemma (^{Miller, 2015}). In a final volume of 40 µl of SAR medium, with 5x10⁶ spermatozoa, 10 µl of WGA-FICT or PNA-FITC was added at a concentration of 15 mg/ml. They were incubated at 25 ºC for 30 minutes, these spermatozoa covering them with light; immediately, preparations were made on object slides, to be observed under the microscope. Each preparation was observed directly under a fluorescence microscope at 260 nm excitation and> 560 nm emission, counting 100 spermatozoa. The presence of membrane carbohydrates was determined by fluorescence patterns and sperm proportion with each determined pattern (^{Naofumi, 2015}).

Statistical analysis

The frequency of live sperm was determined, with normal morphology and with the different fluorescence patterns in the fresh and thawed samples, which were expressed as a proportion with their respective standard error (SE). The different variables were compared between the groups of fresh and thawed spermatozoa with a Xi² test, with an alpha of 0.05; using the free access statistical package ^{EpiInfo 7.3.}

Sperm evaluation parameters

The percentages of live sperm decreased (P <0.05), approximately 30% in post-thaw; finding averages of 89% in fresh sperm and 58% post-thaw. Live sperm percentages that were determined in releases with different spermatophore numbers showed differences (P <0.05), finding a range of 79% to 100% in fresh sperm and from 45% to 67% in thawed sperm (Table 1).

Similarly, normal sperm morphology showed a reduction (P <0.05) of approximately 15%, finding percentages of 98% in normal fresh sperm morphology and 83% post-thaw. Spermatozoa percentages with normal morphology that were determined in releases with different spermatophore numbers did not show differences (P> 0.05), finding an average range of 95% to 100% in fresh spermatozoa. However, certain post-thaw was different (P<0.5), with percentages with an average range between 78% and 90% (Table 1).

Presence and distribution of membrane carbohydrates

With the use of the WGA-FITC lectin: In fresh and post-thaw sperm, the fluorescence intensity emitted by the WGA-FITC lectin on the sperm membrane, evidenced the presence of N-Acetyl glucosamine residues present in the spermatic membrane of A mexicanum. Two fluorescence patterns were determined called: A pattern) with homogeneous intense fluorescence in the flagellum and neck region and with less intensity, but evident in the head one (Figure 1A); and B pattern), with evident homogeneous fluorescence throughout the sperm structure (Figure 1 B).

Figure. 1 Sperm fluorescence patterns with WGA-FITC lectin. 1A). Higher fluorescence intensity is observed in the flagellum and neck regions and with less intensity in the head. 1B is observed with homogeneous intensity throughout the spermatic structure 

The proportion of patterns determined with WGA-FITC (table 2), showed that:

The percentage of sperm with A pattern, from releases of twelve to six spermatophores, were similar (P> 0.05) fresh and post-thaw; in comparison with the percentages of sperm from releases between eight and three spermatophores, in which it increased (P <0.05), in thawed sperm. When comparing the percentages of sperm with pattern A, obtained in fresh sperm from each specimen, an average of 48% was determined, with a range between 44% and 56%; without finding difference (P <0.05) between these, in thawed spermatozoa. An average of 54% was determined with a range between 46% to 57%, finding higher percentages (P> 0.05), in sperm with pattern A, from releases with 3 and 4 spermatophores.

The percentages of sperm with B pattern, from releases of twelve to six spermatophores, were similar (P> 0.05) fresh and post-thaw; the percentages of sperm from releases with four and three spermatophores were higher (P <0.05) when fresh, compared to those thawed. In fresh, an average of 53% was determined, with a range between 46 to 65%, without finding a difference (P <0.05) between them; in thawed sperm, an average of 46% was determined with a range between 42% to 53%, finding higher percentages (P> 0.05) in sperm from releases with 10 and 12 spermatophores.

When carrying out the total general comparison (Pool) of the percentages, a difference was evident and inversely the difference by total percentages determined, which were for sperm with A pattern: Xi ² of 7.21 p <0.05, in fresh semen with 47.7% and 54.3% for thawed semen. Regarding sperm with B pattern: a Xi ² of 10.8 p <0.05 was determined, in fresh semen with 53.5% and 45.6% in thawed semen.

With the use of the PNA-FITC lectin: in fresh and post-thaw sperm, the intensity of the fluorescence emitted by the PNA-FITC lectin on the sperm membrane, which evidenced the presence of β-galactose glycosidic residues, present in the sperm membrane of A mexicanum. Two fluorescence patterns were determined: C) with intense and homogeneous fluorescence throughout the entire sperm structure (Figure 2A); pattern D) with homogeneous faint fluorescence throughout the sperm structure (Figure 2B).

Figure 2 Fluorescence patterns obtained with PNA-FITC lectin: A) With intense homogeneous fluorescence, B) With homogeneous faint fluorescence 

The proportion of patterns determined with PNA-FITC, (Table 3), showed that:

With C and D patterns, sperm percentages from releases with 6 to 12 spermatophores were similar (P> 0.05) fresh and post-thaw, compared to sperm percentages from releases with three and four spermatophores, which were increased (P <0.05), in thawed sperm.

Spermatozoa percentages with C and D pattern, determined with the lectin PNA-FITC, only showed a difference (P <0.05) after thawing, when the spermatozoa came from releases with eight spermatophores, observing a higher percentage in spermatozoa with C pattern post thawing and conversely, in sperm with D pattern. The percentage was higher in fresh semen, in the percentages of sperm from releases with twelve, ten, six, four or three spermatophores, When comparing the percentages of sperm with C pattern of each specimen, fresh with an average of 54%, with a range between 49% to 58 % and post thawing with an average of 60% and with a range between 47% to 69%, no differences were found (P <0.05).

When comparing the percentages of sperm with D pattern of each specimen, fresh with an average of 48%, with a range between 42% to 55%, no different percentages were observed (P <0.05). In thawed spermatozoa, an average of 42% was found, with a range 24% to 53%, no difference percentages were observed (P <0.05).