Sperm Morphophysiology in Different Sections of the Rooster Reproductive Tract/Morfofisiologia Espermatica en Diferentes Secciones del Tracto Reproductivo del Gallo.
INTRODUCTIONA specialized region or structure similar to epididymis in mammals that participates on sperm cell nutrition and maturation processes is lacking on the rooster reproductive tract. So it would be relevant to specify which structure of the vas deferens are subjected to a maturation process before ejaculation. Either sperm cell maturation occurs in the female reproductive system or, it is not needed, as it has been reported that a sperm sample obtained from the testicle may fertilize oocytes in vivo (Howarth, 1983). Sperm storage tubules (SST) have been found on the hen reproductive tract and they are located at the utero-vaginal junction in the oviduct (Holt, 2011; Sasanami et al., 2012). Rooster sperm cells are able to survive between two and fifteen weeks in the SST (Bakst, 2011).
A consensus has not been reached regarding the site where capacitation and acrosome reaction occur, i.e. whether in females or males (Lemoine et al., 2009 & Ahammad et al., 2011a). Biochemical changes and modifications to cell membrane fluidity occur during capacitation of mammalian sperm, in addition to intercellular pH shifts, increased permeability to [Ca.sup.2+] ions, modification of protein phosphorylation patterns and lipid composition (Albarracin et al., 2004). Sperm cell capacitation is a prerequisite for the acrosome reaction in mammalian sperm in order to attain their fertilizing ability (Jones et al., 2007). However, it is not clear if a capacitation process similar to that in mammals is needed before the rooster sperm cells attain their fertilizing ability (Ahammad et al., 2011b). Sperm cell capacitation was evaluated in Meleagris gallopavo by using chlortetracycline (CTC). Morphophysiological changes were observed because an increase of intracytoplasmic [Ca.sup.2+] (Herrera et al., 2017b). Additionally, the N-Acetylglucosamine and sialic acid exhibited by glycoproteins on the sperm cell membrane are involved in gamete recognition in order to achieve fertilization (Cardona Maya et al., 2005; Hernandez Cruz et al., 2005). Lectin Wheat germ agglutinin (WGA) conjugated with isitiosionate of fluoresceine (WGA-FITC) has been used to identify these carbohydrates in vitro. Intracellular [Ca.sup.2+] distribution and the presence of membrane carbohydrates correlate with both capacitation and acrosome reaction processes and these events are assessed in order to evaluate the sperm's fertilizing ability. Therefore, the morphophysiological changes that sperm cells undergo during both capacitation and acrosome reaction processes were analyzed in this study that includes different regions of the reproductive system and the ejaculate.
MATERIAL AND METHOD
Ethical considerations. In accordance with the NOM-062-ZOO-1999 (Norma Oficial Mexicana, 2011) standard regarding the care and use of laboratory animals, 25 Rhode Island Red roosters were used.
Sperm collection. Ejaculates were obtained by aspiration after performing a dorsoventral massage, once per day (NOM-062-ZOO-1999). Roosters were euthanized according to NOM-033-SAG/ZOO-2014 (Norma Oficial Mexicana, 2015), in order to obtain spermatozoa from the testicle and the different vas deferens regions (cranial, medial, and caudal). Subsequently, they were collected in Eppendorf tubes containing 500 [micro]L of Lake solution, (0.09 M sodium glutamate, 0.04 M fructose, 0.003 M magnesium acetate, and 0.05 M potassium acetate, pH of 7.2, and a 330 mOsm osmolarity). Sperms were collected from the tissues by swim up (Herrera et al., 2005), and they were kept at 5 [degrees]C in Lake solution until further evaluation (Herrera et al., 2017b).
Semen basic evaluation. Sperm concentration was determined by optic microscopy with a Neubauer chamber. Motility was analyzed at 400x. Sperm viability and morphology were evaluated by eosin-nigrosin vital staining (1 % eosin and 5 % nigrosin) using microscope (Olympus BX51) immersion oil at 100X. Two hundred cells were evaluated by preparation (Santiago-Moreno et al., 2011 & Herrera et al., 2017a).
Evaluation of [Ca.sup.2+] distribution. 500 [micro]L-aliquots of Lake solution containing 5x106 sperm cells were incubated. To evidence [Ca.sup.2+] presence and its distribution, 10 [micro]L of a clortetracicline (CTC) solution (0.009 M) was previously incubated during ten minutes at 37 [degrees]C in thermal stage protected from the light (Ochoa et al., 2014 & Herrera et al., 2017a).
Assessment of N- acetylglucosamine distribution. A 10 [micro]L-aliquot of a lectin WGA -FITC solution was incubated as a 1:50 dilution at 37 [degrees]C during thirty minutes protected from the light. It was later centrifuged (2,897 G x 1 min), the supernatant was discarded and the cell pellet was suspended with 5 [micro]L ethanol at 2 [degrees]C until further evaluation. After these aliquots were prepared, they were placed on glass slides to be visualized under a fluorescence microscope at 100X, using excitation and emission wavelengths of 488 nm and 560 nm, respectively (Herrera et al., 2005). For data processing, one hundred sperm cells were counted and the images were evaluated using the Image Pro Plus software, version 6.2.1.
The test conditions were taken into account when the sperm cell sample was obtained and evaluated in a post mortem time period less than 10 min. These are suitable conditions for sperm cell capacitation 10 and also to evaluate their ability to undergo acrosome reaction. The latter was induced by incubating 5x106 sperm cells with 20 [micro]L of perivitelline layer (PVL) and kept for 40 min at 37 [degrees]C (Ahammad et al., 2011b).
Statistical Analysis. A randomized model and a Shapiro-Wilk test were used to evaluate normality, whereas the Levene test was used for homoscedasticity. A variance analysis was performed to evaluate fluorescence pattern changes with CTC and PVL+CTC. When differences occurred, a Tukey test was carried out. Differences regarding the effect by region were estimated with a Kruskal-Wallis test after performing the basic evaluation and the WGA or PVL+WGA-FITC fluorescence patterns. The data was analyzed using the R Studio software (Macintosh; Intel Mac OS X 10_11_1, version 0.99.484-[c] 2009-2015 R Studio, Inc.) with a P<0.05 significance level.
RESULTS
Semen basic evaluation. Sperm cell motility is significantly different between regions, 11.3 % mobility was observed in the testis region and this value increased after ejaculation (90.0 %). The live spermatozoa percentage differed among the different regions: a 91.40 % value was found in sperm samples collected from testis, whereas the highest value (97.86 %) was observed on the caudal region. The highest percentage (94.80 %) of sperm cells possessing normal morphology was found in the caudal region, with significant differences (P<0.05) among regions. Subsequently, taking into account these indicators (motility, live spermatozoa and normal morphology), the sperm cell's ability to undergo biochemical changes was assessed during its passage through the vas deferens until their ejaculation (Table I).
As indicated in Table II, the sperm morphometric indicators showed significant differences (P<0.05) in all regions. A total length of 105.54 mm was observed in ejaculated spermatozoa. Increases in head, neck and tail length were also observed as sperm passed through the vas deferens until their ejaculation.
Three [Ca.sup.2+] distribution patterns were observed: A, B and C. The former (intact sperm cells) was characterized by fluorescence in the head, middle part and its flagellum. The B pattern (sperm cells that underwent capacitation) shows fluorescence in the acrosome and at the-equatorial zone and the middle section. The C pattern (acrosome reaction): Shows low fluorescence in the middle section and the flagellum. The pattern proportion observed on the rooster reproductive tract was modified after performing a coincubation of PVL.
Based on the previous classification, Table III shows the percentage of intact sperm cells that underwent capacitation along with acrosomal reaction in fresh samples and after incubation in presence of PVL. A higher percentage of intact sperm cells was observed after their ejaculation when compared to a 66.71 %. Value in other regions of the reproductive tract. Regarding the percentage of sperm cells undergoing capacitation, a higher percentage (31.57 %) was found at the caudal region whereas this value decreased to 18.14 % after ejaculation. Thus, the caudal region may function as reservoir and, if the sperm cells therein are not ejaculated, they undergo anatomical and biochemical modifications. At the caudal region, the observed percentage values of sperm cells undergoing acrosomal reaction were 28.00 % and 14.57 %. It can be hypothesized that, from the moment sperm cells are produced in testis they undergo important morphophysiological changes when they pass through vas deferens until their subsequent ejaculation. The latter assuming that some specialized cells reside within these ducts that induce such anatomic and physiologic changes in sperm cells. Table III shows a comparison between fresh samples and those incubated with PVL in order to induce an acrosomal reaction. In these conditions, significant differences (P<0.05) were observed regarding intact sperm cells in contrast to those undergoing capacitation at the testicular, cranial, medial, caudal and ejaculated regions. Only the percentage of sperm cells undergoing capacitation did not show significant differences in any region. An increase of sperm cells undergoing acrosome reaction was also observed after incubating with PVL when compared to fresh samples. This finding corroborates the patterns previously observed after both treatments.
Three fluorescence patterns were observed after the WGA-FITC test was performed. Pattern A: uniformly fluorescent sperm cells, implicating an intact membrane. Pattern B, sperm cells exhibiting fluorescence at the acrosome and the middle sections, thus they were classified as undergoing capacitation. Pattern C: sperm cells displaying fluorescence on the head that was greater in intensity at the acrosomal zone, thus they were considered as undergoing acrosomal reaction. Table IV shows the percentage of intact sperm cells in fresh treatment and in those incubated with PVL. Significant differences (P<0.05) were observed when the testicular, cranial, medial and caudal regions were compared. No differences were found on the ejaculate.
Regarding the percentage of sperm cells undergoing capacitation, no significant difference was found (P<0.05) in any of the regions either as fresh or in the presence of PVL. Finally, significant differences were observed when both conditions were compared regarding the percentage of sperm cells exhibiting acrosomal, specifically in the testicular, caudal and medial regions. Thus, the caudal region and those that were ejaculated did not display differences between both conditions. Similarly, the data on Table IV suggest that as sperm cells traverse the vas deferens a decrease of intact spermatozoa occurs whereas those undergoing acrosomal reaction increase. This indicates that sperm cells mature within specific regions located at the vas deferens in which they undergo physiological changes regarding their membrane structure as well as morphological changes.
DISCUSSION
The results obtained in this study showed an increased sperm cell motility from the moment they pass through the vas deferens until they are ejaculated. This is important as the sperm cell is enabled to fertilize the oocyte (Moce et al., 2010). However, this increased sperm cell motility renders them energetic because of their limited mitochondrial content until the moment they attain the hyperactivation movement (Etches, 1996), a particular feature of capacitated sperm (Ochoa et al.). This movement ceases, suggesting the existence of an inactivating process occurring on the chicken SST, as previously pointed (Sasanami et al.). An increased motility at the vas deferens has been in Coturnix japonica (Nixon et al., 2014). They observed a testicular motility value of 20 %, whereas this value was 80 % at the vas deferens caudal section. The values we obtained for rooster in this study was 11.33 % in testis and 78 % at the caudal section.
This finding supports the use of G gallus as an experimental model to collect motile sperm cells from a different bird species in order to be used for breeding or preservation purposes. In this work we also assessed the percentage of live sperm cells in testis (91.40 %) and after ejaculation (96.46 %). These percentage values o were similar to those by others studies (Herrera et al., 2005 & Santiago-Moreno et al.). In vitro fertilization (IVF) requires the highest amount of live spermatozoa, although low concentrations are used to perform oocyte fertilization (Mizushima et al., 2014). The parameters observed in this study regarding sperm cell morphology showed 89.2 % of normal spermatozoa in testis and 91.40 % in the ejaculate. These percentages agree with those obtained in other study they observed 89.8 % of normal morphology in G gallus (Tabatabaei et al., 2009). The Lake medium was used for both viability and morphology assessments of normal sperm cells in this study as it preserved these variables, probably because of its composition, low osmolarity and pH. Similar results were obtained by other authors who used this medium (Umapathy et al., 2005 & Herrera et al., 2005).
Similarly, sperm cell morphometry was found to be relevant as maturation and anatomical changes were evidenced when they pass from the testis to the vas deferens to be finally ejaculated. An average size value of 91.49 mm was measured for spermatozoa in testis that subsequently changed to 105.5 mm after their ejaculation. This result is different from that reported by Long (2006), as they observed an 80-90 mm size in G. gallus sperm cells. Regarding the percentages of fresh capacitated sperm cells and those displaying acrosomal reaction observed after a CTC staining, we found 57.77 % of intact spermatozoa along the reproductive tract until their ejaculation: 25.68 % of them underwent capacitation and 19.28 % displayed an acrosomal reaction. However, these values changed after they were co-incubated with PVL as 18.42 % of intact sperm cells was detected in all sections: 28.65 % of them were capacitated and 51.74 % were characterized by an acrosomal reaction. These results suggest that sperm cells located in G. gallus testis possess a fertilizing ability, as previously demonstrated (Howarth), after performing an intramagnal insemination in hens. Similarly, the hypothesis stating that bird spermatozoa do not need to access the female reproductive tract in order to undergo the processes leading to oocyte fertilization is supported by our results. 6,10 Based on this, it is confirmed that sperm cells collected from different sections of the male reproductive tract may be used by different techniques of assisted reproduction in order to preserve endangered birds or those possessing a genetic and commercial value. This, based on the fact that a high percentage of intact spermatozoa is obtained that may be able to withstand cryopreservation if required, but also on the amount of capacitated sperm cells as well as those displaying acrosomal reaction to be used for IVF or artificial insemination.
Regarding the WGA-FITC lectin staining, N-acetyl-glucosamine was identified in rooster spermatozoa and the observed patterns indicate its distribution. When intact spermatozoa displayed fluorescence in its entire structure indicated that N-acetyl-glucosamine receptors are still internalized within the plasma membrane (Cardona Maya et al.). Spermatozoa displaying fluorescence at the acrosome and in the post-equatorial zone were identified as capacitated. Cholesterol levels on the spermatozoon plasma membrane regulate the abundance of these carbohydrate receptors because when capacitation occurs a cholesterol increase is observed caused by the presence of its acceptor molecules. This promotes the suitable conditions for receptor externalization to the outer plasma membrane and finally those spermatozoa with the appropriate physiological state undergo a reaction exhibiting fluorescence only in their heads. In this location the receptors are already externalized creating a spermatozoid-oocyte recognition site and thus the fertilization process is enabled. Altogether, our results indicate that rooster spermatozoa do not need a reservoir site to perform carbohydrate incorporation in order to create a spermatozoa-oocyte recognition site. This proposal is based on the fact that spermatozoa display N-acetyl-glucosamine receptors when they are located in testis, indicating that they already possess a fertilizing ability. This capacity increases as they traverse the male's reproductive tract. Thus, we propose that spermatozoa may be collected from different regions of reproductive tract when they are to be of use for bird breeding techniques, for commercial purposes or in a broader field of opportunity such as the preservation of endangered wild species.
CONCLUSION
Spermatozoa obtained from different regions of the reproductive tract of the rooster demonstrate in vitro an acrosome reaction capacity without requiring a precondition of sperm capacitation, which may be related to its fertilizing capacity. This may be associated with its fertilization ability in vivo. Therefore, the sperm collected from different sections of the rooster reproductive tract may be used in different techniques of assisted reproduction without requiring a previous sperm capacitation.
ACKNOWLEDGMENTS: To the Consejo Nacional de Ciencia y Tecnologia (CONACyT) Mexico, for scholarships 417462 to JAGS.
GONZALEZ-SANTOS, J. A.; AVALOS-RODRIGUEZ, A.; MARTINEZ-GARCIA, J. A.; ROSALES-TORRES, A. M. & HERRERA-BARRAGAN, J. A. Morfofisiologia espermatica en diferentes secciones del tracto reproductivo del gallo. Int. J. Morphol., 37(3):861-866, 2019.
RESUMEN: Es importante conocer los cambios morfologicos que se producen en los espermatozoides del gallo durante su paso por el tracto reproductivo y que ayudan a comprender como adquieren su capacidad de fertilizacion. Se analizaron cambios morfofisiologicos relacionados con los procesos de capacitacion y reaccion acrosomal de los espermatozoides presentes en los diferentes segmentos del tracto reproductor del gallo. Se obtuvieron espermatozoides de diferentes regiones del tracto reproductor del gallo y de espermatozoides de eyaculado. Se usaron 25 gallos Rhode Island Red con fertilidad probada. Se realizaron evaluaciones basicas, con clortetraciclina (CTC) y lectina Wheat germ agglutinin conjugada con isotiosionato de fluoresceina (WGA-FITC) para determinar los parametros morfofisiologicos. La motilidad del esperma aumenta (P<0,05) durante el paso de los espermatozoides desde el testiculo hasta que son eyaculados. Los parametros de viabilidad y morfologia tambien muestran diferencias (P <0,05) en los diferentes segmentos del tracto. La morfometria mostro una contraccion de los espermatozoides (P<0,05) en los segmentos craneal y medial del conducto deferente. La capacidad de reaccion acrosomal evaluada con clortetraciclina CTC o WGA-FITC, fue evidente al aumentar los parametros (P<0,05) con el uso de membrana perivitelina en los espermatozoides del tracto reproductivo y del eyaculado. los espermatozoides del tracto reproductivo del gallo demuestran capacidad de reaccion acrosomal sin requerir una condicion previa de capacitacion espermatica. Por otro lado, no muestran parametros de descapacitacion espermatica lo que implica que no pueden almacenar en ningun segmento del tracto reproductivo.
PALABRAS CLAVE: Acrosoma; Carbohidratos; Membrana; Conducto deferente.
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Jorge A. Gonzalez-Santos (1); Alejandro Avalos-Rodriguez (2); Jose A. Martinez-Garcia (2); Ana M. Rosales-Torres (2) & Jose A. Herrera-Barragan (2)
(1) Doctorado en Ciencias Agropecuarias, Universidad Autonoma Metropolitana-Xochimilco, Mexico.
(2) Departamento de Produccion Agricola y Animal; Universidad Autonoma Metropolitana-Xochimilco, Mexico.
Corresponding author:
Jose Antonio Herrera Barragan:
Calzada del Hueso 1100
Col. Villa Quietud
Delegacion Coyoacan
C.P. 04960
MEXICO
Email: herreraaves@gmail.com
jherrerab@correo.xoc.uam.mx
Received: 20-10-2018
Accepted: 04-02-2019
GONZALEZ-SANTOS, J. A.; AVALOS-RODRIGUEZ, A.; MARTINEZ-GARCIA, J. A.; ROSALES-TORRES, A. M. & HERRERA-BARRAGAN, J. A. Sperm morphophysiology in different sections of the rooster reproductive tract. Int J. Morphol., 37(3):861-866, 2019.
Table I. Evaluation seminal basic in different sections of the vas
deferens and ejaculate.
Sperm cell Testicle Cranial Vas deferens Caudal Ejaculate
Indicator Medial
Motility (%) 11.33e 56.33d 73.00c 78.00b 90.00a
Viability (%) 91.40c 93.93bc 95.66ba 97.86a 96.46ba
Morphology (%) 89.26bc 84.60c 90.86ba 94.80a 91.40ba
Sperm cell SEM
Indicator
Motility (%) 1.08
Viability (%) 0.79
Morphology (%) 1.28
Different letters on different region rows represent significant
differences (P< 0.05). Values are expressed as mean and SEM (n=25).
Table II. Length of the sperm from vas deferens and ejaculate of the
rooster.
Sperm Indicator Testicle Vas deferens
(n=15)
Craneal Medial Caudal
Head length 17.56a 16.49a 15.84a 16.34a
([micro]m)
Midpiece ([micro]m) 4.73a 4.57 (ba) 4.01b (c) 3.92c
Tail length 69.19b (a) 55.21b 67.74 (b) 70.68 (ba)
([micro]m)
Total length 91.49b (a) 76.29b 87.60 (b) 90.95 (ba)
([micro]m)
Sperm Indicator Ejaculate SEM
(n=15)
Head length 16.36a 0.47
([micro]m)
Midpiece ([micro]m) 4.17 (bac) 0.15
Tail length 85.00a 4.08
([micro]m)
Total length 105.54 (a) 3.96
([micro]m)
Different letters on different region rows represent significant
differences (P< 0.05). Values are expressed as mean and SEM.
Table III. Parameters sperm of capacitation an acrosome reaction in
vitro in the presence of PVL as measured with CTC.
Zone Treatment Intact Sperm cells (%) Reaction
Capacitated
Testicle Control 62.57a 24.14 14.57a
(n=25) PVL 19.57b 30.57 45.00b
SEM 5.39 3.10 4.36
Craneal Control 54.71a 28.43 19.00a
(n=25) PVL 22.28b 33.57 43.57b
SEM 3.71 3.60 4.60
Vas Medial Control 59.43a 26.14 18.71a
deferens (n=25) PVL 21.57b 25.29 52.29b
SEM 3.68 2.70 3.23
Caudal Control 45.43a 31.57 28.00a
(n=25) PVL 16.14b 27.00 57.29b
SEM 5.46 2.22 3.28
Ejaculate Control 66.71a 18.14a 16.14a
(n=25) PVL 12.571 (b) 26.86b 60.57b
SEM 2.58 2.72 3.20
Letters distinct on different treatment represent significant
differences (P<0.05). Values are expressed as mean and standard error
mean (SEM). Perivitelline layer (PVL). Clortetracicline (CTC).
Table IV. Parameters sperm of capacitation an acrosome reaction in
vitro in the presence of PVL as measured with WGA-FITC.
Zone Treatment Intact (%) Sperm cells Reaction
Capacitated (%) (%)
Testicle Control 44.71a 23.86 23.57 (a)
(n=15) PVL 14.86b 18.29 55.71b
SEM 3.52 3.25 6.99
Craneal Control 49.49a 29.00 13.86 (a)
(n=15) PVL 13.71b 29.43 55.29b
EEM 3.46 4.99 3.77
Vas Medial Control 41.00a 27.71 10.14 (a)
deferens (n=15) PVL 16.57b 35.29 56.14b
SEM 5.941 5.88 4.11
Caudal Control 13.29a 25.71 47.71
(n=15) PVL 20.86b 23.71 57.29
SEM 2.15 5.29 6.00
Ejaculate Control 18.14 26.14 57.14
(n=25) PVL 16.29 31.57 52.00
SEM 2.29 5.30 5.01
Letters distinct on different treatment represent significant
differences (P<0.05). Values are expressed as mean and standard
error mean (SEM). Perivitelline layer (PVL). Wheat Germ Agglutinin
lectin (WGA).
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