Characteristics of ejaculates and spermatozoa in snakes

1. Introduction

Mexico is a mega diverse country –it has 393 snake groups (Paredes-García et al., 2011; Flores-Villela y García-Vázquez, 2014) and such abundance has long been a part of the pre-Hispanic cultures cosmovision, particularly the rattlesnake, known as the god Quetzalcoatl by the Aztecs, represented as a snake with a halo of feathers, a wavy feather-covered body, and its tail with the typical rings (Bueno, 2000), and as Kukulkan by the Mayans, embodied in the pyramid of Chichen Itza, where during the spring equinox sunlight illuminates the stairway forming the body of the snake that descends to the base of the pyramid, where it joins the head of a rattlesnake (Ojeda, 2004). On the facade of the governor’s palace in Uxmal, rests what may be considered the first model of natural geometry, which uses the diamonds from the skin of the rattlesnake as a pattern; for the Mayans this rhombus meant mathematical certainty, hence the concept of a square sky, the four phases of the moon and the cardinal points (Bueno, 2000). Within the Olmec, Mayan, Zapotec and Aztec iconography and architecture, as well as in the national emblem, the snake symbolized is indeed from the Crotalinae genus (Fuentes-Mascorro, 2014).

According to Gómez et al. (2005), capsules that supposedly contain rattlesnake meat, and the meat itself, are sold without any sanitary control or record of its origin, promising that they will aid the patients fighting AIDS and cancer. Camacho-Escobar et al. (n. d.) indicate that in the coast of Oaxaca the Crotalus aquilus meat, ground and dissolved in water, is used as a treatment for the Newcastle disease in Meleagris gallopavo turkeys. Plus, every year, in line with the World Health Organization, 5.4 million snake bites are registered, causing from 1.8 to 2.7 million cases of poisoning and between 81,410 and 137,880 deaths (Siria and Arellano, 2009). Therefore, poisonous snakes are hunted down for their meat and because of their interaction with humans; this has placed the ophidian accident as a public health issue, enlisting some snake species as subject to protection or threatened, as stated by the Official Mexican Standard 059-SEMARNAT-2010.

The increase of herpetophilia in recent years has caused the distribution of species far from the places where they originally live, raising the need to produce fabotherapics, not only for species of local distribution, but now as well for others that travel around the world through legal markets and illegal trade. The confiscation and guarding of these specimens facilitate obtaining the biological material necessary to produce antidotes, and to isolate and study the proteins for their possible use as drugs, something that takes on a new and interesting meaning since the publication of the studies by Casewell et al. (2012), in which the nesting of genes coding for proteins with physiological roles within the clades of venom toxins was found.

The Animal Reproduction Research Laboratory (ARRL) at the Autonomous University of Oaxaca Benito Juárez, in collaboration with the Herpetario Reptilium and the Zacango Zoo, has recently kept a line of research focused on the application of assisted reproduction techniques in reptiles, first focused on semen conservation, for a later use in the artificial insemination of females far from the male specimen. So in this paper the breakthroughs made are presented.

2. Semen extraction technique

(Fuentes-Mascorro and Álvarez, 2016)

Once the mating behavior was observed in viperid and colubrid elapids, a technique was designed to allow the semen extraction in specimens of said three subclasses. It basically consists of the containment of the specimen by trained personnel; for safety reasons, for both the restrainer and the specimen, the snake is handled in a transparent herpetological tube where the animal introduces the anterior third of its body, ensuring the non-return of the specimen. The technician performing the extraction proceeds to give a ventral cranial-caudal massage in the distal third of the snake’s body, and a circular massage in the area of the cloacal sphincter and in the access sphincters of the hemipenes sacs; once the snake relaxes its sphincters, a diluent is introduced at 28° C in the cloaca and the sacs, then pressure is applied on the region so that the specimen expels the liquid (the liquid from the cloaca does not always return, sometimes the snake retains it or absorbs it); the excess liquid is removed with soft tissue paper. In specimens that are or have been in sexual activity, the male expels a gelatinous material from the hemipenes sacs and then a fluid containing spermatozoa –its consistency goes from pasty to liquid, and the spermatozoa present in it do not always have movement. Using this technique in specimens with sexual activity, up to 30 imprints have been obtained; it should be noted that it is not necessary for the specimen to evert the hemipenes to ejaculate.

It is very likely that the gelatinous material coming out of the hemipenes sacs corresponds to semen ejaculated despite the absence of the female, that passes through the set of grooves that communicate the cloaca with the hemipenes sacs. This material can be white, yellowish, even grayish if it has been there for some time; it can be found at the entrance of the hemipenes sacs, dry and adhered to the specimen’s skin; if so, it is recommended to remove it by moistening it with warm liquid to avoid eroding the skin.

3. Macroscopic evaluation

The snake ejaculation is emitted in waves. When a male and a female are copulating, there is a phase of stimulation; in Crotalinae, the female stays still and the male advances crawling on the female’s body once it has introduced the hemipenis into the female cloaca, making a lateral movement with the head to both sides; when it reaches the height of the female head, it draws back its body and ejaculates, and then initiates another stimulation session –a copulation can last up to 18 hours. When the semen is removed from the males, the specimen is stimulated simulating the sensation that it would have crawling on the female, and so it ejaculates a drop of semen, which can go from translucent to milky white, with a consistency from liquid to mucous, of a very low volume; then a new stimulation session is carried out and so on. In colubrid specimens, the copulation is performed as they interlace each other from the middle third to the posterior third: the male introduces the hemipenis into the female cloaca and slides its body intertwining it with the female to complete a "nuptial hug", ejaculating soon after; then it relaxes the body that stays interlaced, and stimulation is repeated –under this system it has been possible to obtain up to 20 ejaculations per session; the sperm is translucent, and 250 μL per ejaculation have been obtained at most; when these specimens are not in breeding season, they do not ejaculate.

In most cases, the ejaculates are collected by imprint on a slide tempered at 28° C; it’s confirmed by observation under the microscope that the imprint contains spermatozoa, 10μL of the diluent that is being used in the foil are poured and mixed with a tip, and the diluted ejaculate is transferred to an Eppendorf tube for further processing. It should be noted that when the ejaculate is thick it does not coagulate like that of humans, so if it is not diluted, it dries up, and this happens too if the imprint does not dissolve quickly. Colubrids, viperid and elapid, can ejaculate spermatozoa without movement, alive at eosin-nigrosin staining; it has been observed that once diluted, spermatozoa begin to show movement and can come out indistinctly in the ejaculate, with and without movement, without any pattern. The pH has been determined using test strips, obtaining a 7.0 value in all specimens, this data should be taken with reservation because the test strips are not very sensitive and the pH scale is logarithmic.

4. Microscopic evaluation

Mass sperm motility is the way spermatozoa move as a group; the microscope slide is placed under the lenses and focused with the 10X objective; a 5 is assigned when the spermatozoa move in groups, forming vigorous whirlpools, similar to those of a moving shoal; 4 to eddies with less vigor; 3 if there are no swirls, but there is group movement; 2 when there is movement in small spermatozoa groups; 1 if the movement is reduced to just a few spermatozoa, and 0 when there is no movement. Under this scale the ejaculates of animals in the middle of breeding season show a 5 mass sperm motility.

A cooperative movement was found in the Crotalus atrox spermatozoa, consisting of groups of hundreds of spermatozoa that coordinate their flagellar motion in order to move in the same direction (Fuentes-Mascorro et al., 2013); this phenomenon has been reported in Parachauliodes japonicus (Hayashi, 1998), and in Rattus norvegicus (Pizzari and Foster, 2008). The recorded videos contain similar images (Figure 1) to those in the paper by Moore et al. (2002).

Figure 1. C. atrox spermatozoa trapped in desquamation tissue, synchronizing the beat of their flagella to move in a coordinated manner and to increase the movement effect; the darkest part corresponds to the  set of 40X heads.

Morphology: the reptilian spermatozoon has a characteristic shape (Figure 2), distinguishing a head and a flagellum; the acrosome that ends in a tip is located in the anterior part of the head, inclined to the right or left. The nucleus that, along with the acrosome, shows the shape of a pyramid is located before the implantation pit; it is continued with a very long middle part and ends in a tail that seems to have no membrane, as in mammalian spermatozoa. Gathering information on abnormalities observed over four years analyzing snake ejaculates, it has been found that out of a 100% of abnormalities the head presents 21.76%, the flagellum 30.57%, a bent and swollen head (with two or more abnormalities) 47.67%. And in the flagellum it was observed a coiled, curled shape, folded on itself, plus angular deviations in waves and the presence of two tails, some with a swollen head and a bent tail –we show some of the abnormalities found in Figures 3 to 8.

Figure 2. Crotalus spp. spermatozoon; the head shows the blue dyed nucleus and the translucent acrosome, the flagellum with an extremely long half piece, and an apparently naked final tail 40X.

Figure 3. C. molossus nigrescens spermatozoa stained with 40X basic fuchsin, the absence of heads is noticeable.

Figure 4. C. molossus oaxacus spermatozoa stained with 40X basic fuchsin. The blue arrows indicate defects in the implantation pit.

Figure 5. C. ravus ravus spermatozoa stained with 40X hematoxylin. A middle piece folded in the upper part is seen, a folded tail to the left, and a flagella folded on itself below, forming an eight.

Figure 6. Stenorrhina f. spermatozoa. Eosin-nigrosin staining with a 40X filter. Swollen heads are seen in the upper right, as well as sperm clustering; as the sample dries they tend to associate in formation.

Figure 7. C. molossus nigrescens spermatozoa. Eosin-nigrosin staining. The deformed head and the two tails at the end of the flagellum are seen in the lower sperm.

Figure 8. Crotalus spp spermatozoa. Eosin-nigrosin staining at 40X, with a camera close up. A different degree of head swelling is seen.

Figure 9 shows the nucleus, the implantation pit or neck, part of the mitochondrial sheath and the dense fibers of the flagellum; the membrane is noticeably thin and labile to the techniques used for its processing. Figure 10 displays the structure of the middle piece or main piece of the flagellum, with a well-defined plasma membrane, the grouping of mitochondria that characterizes it and the fibrous sheath. In Figure 11 we see the difference between the middle part and the tail, or final piece of the flagellum, which is not surrounded by the cell membrane.

Figure 9. Electronic photomicrography of transmission. The arrow marks a thin, electron-dense, irregular plasma membrane that extends to the middle part. The elongated and electron-dense nucleus (N). The sperm neck (CE). General contrast technique with uranyl acetate and lead citrate. Zoom in 12 000x.

Figure 10. Electronic photomicrography of transmission. Middle part (PM) or main part electron-dense, cell membrane (MC), mitochondria (M), and fibrous sheath (VF). Uranyl contrast and lead citrate technique. Zoom in 7 000x.

Figure 11. (PF) or tail, the axonema is seen surrounded by dense electron-dense external fibers, a cross section of the middle piece (PM), surrounded by mitochondria (M). Contrast technique with uranyl acetate and lead citrate. Zoom in 12 000x.

Figure 12. Spermatozoa. Stained with rhodamine B and propidium iodide, through confocal microscopy we can see the heterogeneity of the heads, the length of the middle part and the integrity of the acrosome despite the swelling of the nucleus, so it could be DNA decondensation.

Figura 13. Fluorescence microscopy; the nucleus is seen in blue color (Hoechst), and the antibody that binds to aquaporin 1 is appreciated in green.

Live-dead. Handling the data of live and dead spermatozoa represented some substantial difficulties with eosin-nigrosin staining used in mammals (Campbell et al., 1956); the snakes spermatozoa began to show some difficulties, as displayed in figures 7 and 8, so that an osmolarity study was carried out with solutions of 200, 300, 400 and 500 mOsm/L, in Crotalus molossus, Crotalus atrox and Crotalus culminatus –osmotic stress was found in all solutions, no differences between species, noting that spermatozoa respond to osmolarities of 200, 400 and 500 mOsm/L, increasing the volume of the region corresponding the nucleus, keeping the acrosome intact (Simón-Salvador et al., 2016).

The osmolarity of Boa constrictor, Morelia spilota, Pituophis deppei, Crotalus culminatus and Crotalus molossus spermatozoa, using solutions of Tyrode modified at 420, 440, 460 and 480 mOsm/L, evaluating at 0, 12, 24 and 36 hours of conservation at 28° C, the percentage of swelling (PS) in the spermatozoa was measured, the results showed for Pituophis deppei 460 mOsm/L from 0 to 48 hours, the spermatozoa was preserved in perfect condition; Boa constrictor 420 mOsm/L showed 0 PS, for 0, 12 and 48 hours; Morelia spilota presents the best results at 420 and 460 mOsm/L; Crotalus culminatus has the lowest percentages of swelling (2 to 40%), between 460 and 480 mOsm/L; and for Crotalus molossus the four osmolarities seem adequate, since the PS range goes from 0 to 49; the osmolarity of the appropriate solution depends on the time required to preserve the sample (Fuentes-Mascorro et al., 2015).

The snake spermatozoa respond with swelling of the head, both to hypoosmotic and to hyperosmotic stress, for which the distribution of aquaporin is currently being sought; in figure 11, the presence of aquaporin 1 in the acrosome region is appreciated, because these proteins are able to allow the entry or exit of water against the osmotic gradient.

Dilutions. To keep the spermatozoa moving at 28°C (cloacal temperature in same genus females), the efficiency of diluents routinely used to conserve mammalian sperm was evaluated; Crotalus genus semen was extracted from the atrox, basiliscus, culminatus, m. nigrescens, m. oaxacus, ravus, simus and scutulatus species; the ejaculates were diluted in 10 μL of the corresponding solution, kept in Eppendorf tubes, in dry bath, and mass motility was evaluated every 30 minutes, using the aforementioned scale from 0 to 5, Table 1 (Fuentes-Mascorro et al., 2014); the longest time of sperm motility conservation was presented with the modified Tyrode medium, which is constituted by the commercial Tyrode supplemented with bovine albumin 5 mg/mL.

Table 1. Mass motility.

Mass motility evaluated with a scale from 0 to 5. In the upper row the diluent is indicated and the time in which the evaluation was finished, the number left of the diagonal line indicates the initial motility, the number on the right side is the final motility in the time indicated in the first row.

Measurement of the spermatozoa. We worked with three free-living Stenorrhina freminvillei specimens weighting 42 grams and with a size of 42.5 cm in total length, Coluber mentovarious weighing 400 grams and with a size of 107.7 centimeters, and Boa constrictor with a weight of 2,400 grams and a size of 157 centimeters, which were captured by interaction with humans; plus a fourth specimen: the Crotalus molossus oaxacus –obtained from the Herpetario Reptilium, a specimen of 750 grams, in captivity with a constant temperature of 25°C and a humidity of 47 to 63%; 5 ejaculates per specimen were obtained for evaluation, 200 spermatozoa were measured per specimen with the motic 2.0 program, the results are shown in Table 2.

Table 2. Evaluation and measurements of the spermatozoa.

Five ejaculates were used per specimen. The morpho-spermatic index is the ratio of the length of the flagella between the length of the head; %=percentage; spz=spermatozoa.

The Boa specimen was at the end of the breeding season. The morpho-spermatic indexes show high results for small-headed spermatozoa, as is the case of the Stenorrhina, the smallest adult specimen, finding no relation between the expected adult length of the genera evaluated and that of the spermatozoa they produce.

5. Conclusion

The snake ejaculate is presented in ejaculatory waves, accompanied by a stimulation phase that continues with the spermatozoa expulsion, which is expelled in very small volumes with a variety of consistency from liquid to mucous, from translucent to milky white. Because the hemipenes do not have an ejaculatory function, their eversion is not necessary, since the ejaculate is guided through the middle groove of the hemipenia. The evaluation of the ejaculate by routine techniques is possible by adjusting the osmolarity corresponding to each genus and species. The reptilian spermatozoa have a characteristic preserved shape, and keeping the appropriate conditions of osmolarity, it is possible to keep it viable in dilution to be transported.