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| felinia 22 |
on line desde enero 2002 |
noviembre 2003 |
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. En el Annual Meeting de TICA se aprobó la aceptación de las siguientes razas a efectos de registro ("Registration Only"): Asian Longhair, Asian Shorthair, Australian Mist, Burmilla, Ceylon, European Shorthair, European Burmese, German Rex, Javanese (registrado como BA, Balinés Longhair), Kurilian Bobtail, Mandarin (registrado como OL, Oriental Longhair), Mandalay, Ragamuffin, Sokoke y Thai. En total, 15 nuevas razas que empiezan el proceso de reconocimiento completo. De momento, su clasificación como "Regitration only" les permite obtener pedigrees pero no participar en Campeonato.
Introducción
La evaluación de semen en el macho reproductor es un método
complementario de rutina en la evaluación del gato infértil o con
enfermedad del tracto reproductivo, así como en la criopreservación de
semen. En
el gato la recolección de semen puede realizarse mediante vagina
artificial o electroeyaculación. El eyaculado obtenido puede utilizarse
para evaluar la calidad de semen de un reproductor, criopreservación de
semen o inseminación artificial (IA). La recolección de semen a partir
de la vagina de la hembra luego del servicio natural, permite obtener
algunos datos sobre el semen de un reproductor. Si bien esta información
puede ser útil es escasa en comparación con la proporcionada por la
contrastación de un eyaculado obtenido mediante vagina artificial o
electroeyaculación. La recolección de espermatozoides de la cola del
epidídimo es un método implementado para la criopreservación de semen
pos mortem.
La
criopreservación de semen e inseminación artificial en el gato doméstico
pueden utilizarse como reservorio genético y posibilidad de reproducción
asistida, así como modelo experimental para felinos silvestres en vías
de extinción. Los bancos de semen reducen la necesidad de machos en un
plantel reproductivo y permiten preservar material genético que de otra
forma se perdería. Así mismo el estudio de la reproducción de los gatos
domésticos constituye un modelo experimental sumamente útil para el
estudio e investigación de especies silvestres. Recolección
de semen
La electroeyaculación es el método más utilizado para la recolección
de semen, sin embargo la vagina artificial puede utilizarse en algunos
gatos entrenados. Otros métodos pueden ser útiles cuando no es posible
aplicar los anteriormente mencionadas. Obtención
de semen mediante vagina artificial
Puede fabricarse una vagina artificial, seccionando y adaptando una
pera de goma pequeña (como las que acoplamos a las pipetas de 1 o 2
mililitros) a un ependorf o a un tubo de pequeño calibre (15). Foto 1 y 2.
La vagina debe precalentarse a 36ºC, colocarse cuando el macho realiza la
monta de la hembra en celo y colectar así el eyaculado. La colecta puede
realizarse sin utilizar una hembra en celo, previo entrenamiento del macho
a la eyaculación mediante manipulación. Sin embargo es preciso recordar
que cualquiera sea el método elegido, el animal requiere un entrenamiento
previo y solo un pequeño porcentaje de los animales logra eyacular
mediante la utilización de vagina artificial (2). Obtención
de semen mediante electroeyaculación
La
electroeyaculación permite obtener semen de todos los animales que posean
la vía neurológica implicada intacta. Sin embargo, una limitante es la
necesidad de someter al animal a una anestesia general a fin de evitar las
molestias que este método produce sobre el mismo (6)
Para la
realización de esta técnica debe utilizarse un electroeyaculador, el
cual consta de un vástago (fuente de electrodos) y una fuente de voltaje
(foto 3). El vástago es introducido entre 7 y 9 centímetros en el recto
con los electrodos ubicados hacia ventral. El pene es exteriorizado
y se lo coloca dentro de un tubo. Se necesitan unos 80 estímulos de entre
2 y 5 voltios para lograr un eyaculado. Los estímulos se dividen en tres
series con un descanso de 2 a 3 minutos, entre cada serie. Con cada estímulo
ocurre una extensión rígida de los miembros posteriores, la cual indica
que el estímulo ha sido adecuado (2, 15). Se ha demostrado que el voltaje
de estimulación afecta la osmolalidad y pH del semen obtenido por este método
(9). También se ha comprobado que la electroeyaculación permite obtener
un eyaculado de mayor volumen pero menor concentración espermática que
la vagina artificial (9).
Este método se reserva solo para animales sanos, para los cuales
el procedimiento implica un riesgo mínimo. Obtención
de semen mediante lavaje vaginal post servicio
Si bien este no es el método ideal para realizar una evaluación seminal,
puede utilizarse cuando no es posible realizar una extracción de semen
mediante vagina artificial y es riesgoso someter al animal a un protocolo
anestésico o el propietario no accede al mismo. Es
preciso considerar que esta práctica es engorrosa ya que el macho debe
realizar un servicio e inmediatamente después la hembra debe ser sedada
para implementar un lavaje vaginal, el cual se realizará con solución
salina fisiológica a 37ºC (15). Debe considerarse que los
espermatozoides recolectados por este método pueden sufrir alteraciones
relacionadas con la técnica de toma de muestra utilizada. El lavaje
vaginal con 1 ml de solución fisiológica permite obtener entre 40 X 104
y 10 X106 espermatozoides (2, 15).
Recuperación
de espermatozoides de la vejiga
Se ha reportado que el gato eyacula entre 15 y 90 % de semen en forma retrógrada
en la vejiga urinaria. Es así que la recuperación de espermatozoides de
la vejiga es usado para determinar si el gato produce espermatozoides.
(15) Colección
de semen a partir del epidídimo Pueden
obtenerse espermatozoides de la cola del epidídimo luego de la castración
o pos mortem. El epidídimo debe lavarse con un diluyente de semen para
obtener los espermatozoides que serán posteriormente criopreservados.
Este es un método
útil y eficaz en la preservación de semen de especies silvestres cuando
los animales mueren en zoológicos o reservas (3,4). Evaluación
de semen
Volumen: el volumen del eyaculado es
pequeño y varía según el método utilizado para su obtención. Cuando
la recolección se realiza con vagina artificial el volumen puede alcanzar
0,12 ml, mientras que con electroeyaculación pueden obtenerse hasta 0,74
ml. El volumen eyaculado puede medirse utilizando micropipetas (9)
Color:
el color es blanquecino, pudiendo ser amarillo si se contamina con
orina y rosado o rojizo si se contamina con sangre (10)
Concentración espermática: puede
oscilar de 13 X106 a
153 X106 espermatozoides totales.
La concentración espermática es mayor cuando se colecta la
muestra con vagina artificial en comparación con las muestras obtenidas
por electroeyaculación (1, 21). Los métodos de conteo son los usados
rutinariamente en la contrastación de semen (cámara de Neubauer, cámara
de Burker y contadores celulares) (2)
Motilidad individual: el porcentaje de
motilidad progresiva se estima en platina térmica, a través de la
observación de una gota de semen puro a 400 X. Se ha comunicado como
normal una motilidad progresiva de entre 60 y 90 % (3, 15) Morfología espermáticaEl espermatozoide felino mide aproximadamente 26 um de largo, a diferencia del canino que mide 36 um (1, 2) La
identificación de formas anormales se realiza mediante microscopía de
contraste de fase o mediante tinciones como Diff-Quik. Se ha comunicado
que el semen normal posee alrededor del 70 % formas normales. Las
anormalidades morfológicas que han sido identificadas son: macrocefalia,
microcefalia, cabeza doble, gota citoplasmática proximal y distal,
cabezas sueltas, colas enrolladas. (15). Muchos
factores influencian la morfología de los espermatozoides en el
eyaculado. Se ha establecido como normospérmico al semen felino que posee
más del 60% de espermatozoides normales. No se conoce la relación entre
morfología espermática y fertilidad in vivo en gatos (13, 14)
Osmolalidad: es aproximadamente de 320
mOsm (16, 19)
PH: oscila
entre 6,6 y 8,8 (16, 19)
Fosfatasa alcalina: el
semen felino es rico en fosfatasa alcalina (16, 19)
Microbiología seminal: las bacterias
aeróbicas aisladas a partir de eyaculados provenientes de gatos sanos
incluyen: E coli, Pseudomona
Aeruginosa, Proteus Mirabilis, Klebsiella SP, Streptococo y Stafilococo.
Estas bacterias constituyen la flora saprofita de la uretra distal y
prepucio del macho (6, 12) Criopreservación
de semen felino
El
semen felino puede ser almacenado por cortos períodos (24-48hs) mediante
refrigeración a 4º C o por largos períodos mediante congelación. La
mayoría de los estudios sobre criopreservación de semen han sido
realizados in vitro. Existen muy pocas comunicaciones de preñeces
logradas mediante inseminación artificial con semen refrigerado o
congelado.
Semen
refrigerado: Se han usado, para la
refrigeración de semen felino, diluyentes en base TRIS con el agregado de
yema de huevo, sin embargo pocos estudios han sido realizados sobre los
diferentes diluyentes que pueden usarse en este procedimiento.
La supervivencia espermática y capacidad fecundante del semen refrigerado
han sido estudiadas solo in vitro. Faltan aún pruebas de campo que
comprueben la capacidad fecundante del semen refrigerado a través del
logro de preñeces obtenidas mediante inseminación artificial (15)
Semen
congelado: el semen felino ha sido
congelado con diluyentes compuestos por trealosa, glicerol, yema de huevo
y antibióticos. La congelación seminal se ha realizado en pastillas y
pajuelas, lográndose mejores resultados con estas últimas (1, 2, 20).
Luego de un período de equilibración a 5º C de entre 30 y 40 minutos se
realiza la congelación mediante vapores de nitrógeno líquido y
posterior inmersión de las pajuelas en el mismo. Estudios más profundos
sobre congelación de semen felino son necesarios para optimizar el método
(2, 15) Inseminación
artificial La
inseminación artificial (IA) en los gatos domésticos ha sido
implementada principalmente como parte de proyectos de investigación,
donde el gato es usado como modelo experimental para el estudio de felinos
salvajes. Las técnicas de IA y conservación de semen pueden se una
herramienta valiosa en los programas reproductivos felinos. El semen
congelado puede utilizarse como reservorio de material genético y puede
emplearse luego de que un animal haya sido castrado o haya muerto. El
sitio en que se deposita el semen, el momento en que se realiza la
inseminación y el número de espermatozoides mótiles y viables son
factores importantes que influencian la tasa de concepción (22, 10)
En la hembra
felina la ovulación es inducida por el estímulo coital, el cual
desencadena la liberación de GnRH desde el hipotálamo provocando la
liberación de LH a partir de la pituitaria (11, 18). Múltiples coitos
son necesarios para que exista una alta probabilidad de que la hembra
ovule. Entre 26 y 30 hs luego de la liberación de LH (si las
concentraciones séricas de la misma son suficientes) ocurre la ovulación
de todos los oocitos maduros (10, 15). Se estima que en un pequeño
porcentaje de hembras (35%) ocurre ovulación espontánea (10). En relación
a la fisiología felina, para realizar IA debe inducirse la ovulación.
Este evento fisiológico puede provocarse mediante la administración de
gonadotrofina coriónica humana (hCG), la cual posee actividad LH (2, 18)
Ocurre la ovulación en la mayoría de las hembras, cuando se administran
100 UI de hCG intramuscular el tercer día del estro. Una alternativa es
el uso de 25 ug de GnRH, hormona liberadora de gonadotrofina, por vía
intramuscular (5, 7, 8, 17). Es
importante destacar que se encuentra en amplio desarrollo mediante
continuas investigaciones un protocolo eficiente para la inducción
hormonal del estro y ovulación con mínimos efectos colaterales. Inseminación
artificial con semen fresco
Se han comunicado preñeces con IA vaginal o uterina con semen fresco. El
semen, debido al escaso volumen eyaculado, puede diluirse con solución
fisiológica o un diluyente en base Tris. Esto permite un mejor manejo del
eyaculado, el cual será depositado en la vagina 24 hs después de la
ovulación o en el útero 30 a 50 minutos luego de ocurrida la misma (2,
22) Para
la IA vaginal puede utilizarse una sonda tomcat, la cual será introducida
en la vagina, la mayoría de las veces, sin utilización de
tranquilizante. El semen será depositado en el fondo de la vagina cerca
del cuello del útero (foto 4). Deben calcularse un mínimo de 80 X 106
espermatozoides para la inseminación intravaginal, lo cual es 10 veces más
alto que lo necesario para la IA intrauterina. (23) Inseminación
artificial con semen congelado La
primer comunicación de una preñez con semen congelado en gato se realiza
en 1976 a partir de IA intravaginal (20) La
tasa de preñez esperada utilizando semen congelado mediante IA
intravaginal es aproximadamente de 10 %. La pobre fertilidad observada
cuando utilizamos semen congelado se relaciona con el daño sufrido por
los espermatozoides durante el proceso de congelación y descongelación
(2). El método y lugar de IA son cruciales cuando se utiliza semen
congelado. La IA intrauterina permite obtener tasas de preñez superiores
en comparación la IA vaginal (16) Conclusiones:
La
reproducción felina ha captado la atención de numerosos científicos en
la última década. El creciente interés sobre este área del
conocimiento se relaciona no solo con las implicancias que tiene sobre la
medicina y reproducción del gato doméstico, sino también con la
posibilidad de utilización del Felis catus como modelo para el estudio de felinos silvestres. El
desarrollo de la fecundación in vitro y la transferencia embrionaria ha
impulsado la actualización de conocimientos de la fisiología
reproductiva felina, realizándose importantes descubrimientos lo cual ha
estimulado el desarrollo de la IA y la criopreservación de semen. La
difusión del conocimiento en este área, permitirá al especialista en
medicina felina, incluir en su práctica diaria procedimientos nuevos
relacionados con la reproducción y biotecnología. Bibliografía: 1) Axner, E. (2000) Sperm morfology and maturation in the domestic cat (Felis Silvestris Catus), with special reference to the morfology and function of tthhe epididdymis. Acta Universitatis Agriculturae Sueciae. 2)
Axnér, E. and Linde-Fosberg, C. (1998). Mating and artificial
insemination in Small animal reproduction and neonatology (eds. G.
Simpson, G. C. England and M. Harvey), 105-111. Cheltenham: BSAVA. 3)
Axnér, E.; Linde-Fosberg, C.; Einarsson. S.; (1999).Morphology and
motility of spermatozoa from different regions of the epididymal duct in
the domestic cat. Theriogenology 52: 767-778. 4)
Axnér, E.; Ström-Holst, B.; Linde-Fosberg,
C. (1998) Morphology of spermatozoa in the cauda epididymis before and
after electroejaculation and a comparison with ejaculated spermatozoa in
the domestic cat. Theriogenology. 50: 973-979. 5)
Colby, E. D.(1980). Suppression/induction of estrus in cats. In Current
Veterinary therapy in theriogrnology. (ed. D. A. Morrow), 861-864.
Philadelphia: W. B. Saunders. 6)
Concannon, P.W. and Lein, D. H. (1983) Feline reproduction. In
Current veterinary therapy, 8th ed. (ed. R. W. Kirk), 932-936.
Philadelphia: W. B. Saunders. 7)
Cline, e. m. ; Jennings, L. L.; Sojka, N. J. (1980) Breeding laboratory
cats during artificially induced estrus. Lab. Anim Sci. 30, 1003-1005. 8)
Davidson, A. P. (2000) CVT Update: Infertility in the queen. In Current
Vet Therapy, 8th ed. (ed. J. D. Bonagura), 929-931.
Philadelphia: W. B. Saunders 9) Dooley, M. P. & Pineda, M. H. 1986. Effect of method of collection on seminal characteristics of the domestic cat. Am. J. Vet. Res. 47, 286-292. 10) Feldman, E. and Nelson, R. 1996. Feline reproduction. In canine and feline endocrinology and reproduction, 2nd ed., 741-768. Philadelphia: W. B. Saunders. 11) Goodrowe, K. L.; Howard, J. G.; Schmidt, P. M. and Wildt, D. E. (1989) Reproductive biology of the domestic cat with special reference to endocrinology, sperm function and in-vitro fertilization. J. Reprod. Fertil. Suppl. 39, 73-90. 12)
Herron, M. A.(1977) Feline reproduction. Vet. Clin. North. Am. 7 (4),
715-722. 13) Howad, J. G.; Brown, J. L.; Bush, M.; Wildt, D. E. (1990) Teratospermic and normospermic domestic cats: ejaculate traits, pituitary-gonadal hormones, and improvement of spermatozoal motility and morphology after swim-up processing. J. Androl.; 11: 204-215. 14) Howad, J. G.; Bush, M.; Hall, L.L.; Wildt, D. E. (1984) Morphological abnormalities in spermatozoa of 28 species on non-domestic felids; 10th Int. Congr Anim. Reprod. Artif. Insem. 57-59. 15)
Jhonston, D.J.; Kuztritz, M.V.R.; Olson, P. (2001) Canine and
feline theriogenology. Ed. Saunders. Philadelphia (United States).
16:287-306. 16)
Johnston, S. D.: Osborne, C. A. & Lipowits, A. J. (1988).
Characterizacion of seminal plasma, prostatic fluid, and bulbourethral
gland secretions in the domestic cat. Prooc. 11th Int. Congr.
Anim. Reprod. & A. I. Dublin, IV, 560. 17)
Kaywalee, C. (2001) Studies on cervical patency and catheterization in the
domestic cat (Felis catus) Master.
Faculty of Veterinary Medicine. Uppsala.
18)
Liège, P. (1992). Induction
de l´ovulation et insémination artificielle chez la chatte, in
Reproduction du chien et du chat. ed Dumon, C.; Fontbonne, A. Ed
P.M.C.A.C. p. 265-270. 19)
Melrose, D. R.& Laing, J. A. (1970) The characteristics of normal
semen. In fertility and infertility in domestic animals, 2nd
ed. (ed. J. A. Laing), 128-160. London: Baillière Tindall & Cassel. 20)
Platz, C. C.; Wildt, D. E.& Seager, S. W. J. (1978) Pregnancy in the
domestic cat after artificial insemination with previously frozen
spermatozoa. J. Reprod. Fertil. 52, 279-282. 21)
Schwartz, D.; McDonald, P. D. M. & Heuchel, V. (1981) On the
relationship between the number of spermatozoa and the probability of
conception. Reprod. Nutr. Develop. 21, 979-988. 22)
Sojka, N. J., Jennings, L. L. & Hamner, C. E. (1970). Artificial
insemination in the cat (felis catus) . Lab. Anim. Care 20, 198-204. 23)
Tsutsui, T.; Tanaka, A.; Takagi, Y.; Nakagawa, K.; Fujimoto, Y.; Murai,
M.; Anzai, M.; Hori, T. (2001). Unilateral intrauterine horn insemination
of fresh semen in cats. J. Vet. Med. Sci. 62. . M.C.
Stornelli y M.A. Stornelli son veterianarios, integrantes de la Cátedra
de Reproducción Animal. Laboratorio Central. Fac. Cs. Vet. U.N.L.P. Calle
60 y 118. La Plata (1900). Bs. As. Argentina. Este artículo se publico
inicialmente en la página de AAMeFe (Asociación Argentina de Medicina
Felina).
IntroductionThere are 37 feline species and all of them, with the exception of the domestic cat, are threatened by extinction, as reported by the Convention of International Trade of Endangered Species (CITES 1973). Wild feline species quite often demonstrate poor breeding performance, both in captive and in natural conditions. One of the most important causes of infertility or sub-fertility is decreased genetic diversity, which is caused by inbreeding due to geographical isolation and population contraction (Wildt DE 1990). An improved reproductive performance in non-domestic Felidae is therefore needed and a successful breeding program frequently involves assisted reproduction techniques (ART), such as artificial insemination (AI). The use of AI can reduce difficulties such as natural aggressiveness of these species, male–female behavioral incompatibility, or physical handicaps (Wildt DE 1990). Moreover, it reduces the risks of infectious disease transmission through copulation. With AI it is also possible to transfer semen from captive to wild populations orbetween wild populations geographically separated, which contributes to restoring genetic vigor. AI can be performed with fresh, cooled or cryopreserved semen, the latter providing long-term storage for postponed use. In felids, the first attempts to prolong sperm viability and preserve its fertilizing ability were performed in the domestic cat. Its role is very important as a comparative model for studies aimed at non-domestic feline species, but ART can also be employed in the domestic cat to obtain kittens from valuable cat breeds. The aim of this paper is a review of the results obtained in feline sperm conservation through cooling and freezing. Semen conservationFor assisted breeding purposes, semen can be collected with an artificial vagina (Sojka and Jennings 1970, Sojka et al 1970 and Zambelli and Belluzzi 1998), with an electroejaculator (Seager 1976, Platz and Seager 1978, Dooley et al 1983, Pineda et al 1983, Johnstone 1984, Herron et al 1986 and Howard 1986) and from epididymides and vasa deferentia of isolated testicles (Howard 1986). Conservation protocols can be applied both to ejaculated and to epididymal spermatozoa. The latter has proven to be a precious source of sperm in case of orchiectomized or dead animals. In order to preserve sperm quality, it is important to know physiological characteristics of the semen that are species-specific, as demonstrated by the poor results which were obtained in the past trying to apply to cat sperm the same diluents and procedures developed for cattle. Undiluted fresh cat semen, stored at +37°C, maintains motility in vitro for 60 min. This period can be prolonged up to 140 min if semen is stored at +23°C, and even more if diluted with an appropriate medium (Goodrowe et al 1989). Thus, great importance is attributed to diluent osmolality. The osmolality of cat semen ranges from 290 to 320 mOsm/kg and the ideal diluent should be about the same value. Hypotonic solutions cause swelling and membrane rupture, which are generally more detrimental than the shrinkage caused by hypertonic solutions (Glover and Watson 1985 and Pukazhenthi et al 2002). The presence of seminal plasma negatively affects spermatozoal fertilizing ability in the cat (McLaughling and Hamner 1974), therefore sperm centrifugation at low speed (300 g) and subsequent removal of seminal plasma significantly prolongs spermatozoal survival time. Better results can be achieved applying the swim-up process, which selects spermatozoa on the basis of motility and structural integrity (Howard et al 1990). Semen coolingBoth ejaculated (Glover and Watson 1985, Glover and Watson 1987, Pope et al 1989, and Pukazhenthi et al 1999) and epididymal spermatozoa (Goodrowe and Hay 1993 and Harris et al 2001) can be prepared for the cooling process. It is also possible to cool the testicles and then flush the epididymides and vasa deferentia in order to collect spermatozoa (Goodrowe and Hay 1993 and Hay and Goodrowe 1993). The semen sample is extended with a diluent and subsequently cooled and stored at +5°C. This procedure allows a short-term storage: overnight (Goodrowe and Hay 1993), for 24 h (Glover and Watson 1985, Pope et al 1989 and Pope et al 1991), or sometimes even up to 5–7 days (Glover and Watson 1987 and Harris et al 2001). Recently, (Harris et al 2002)demonstrated that blastocyst development occurs after in vitro fertilization of cat oocytes with spermatozoa stored at +4°C for 14 days. Some studies have focused on cooling diluents with the aim of finding the ideal medium to limit cold-induced damages on spermatozoal motility and morphology. (Glover and Watson 1985) suggested a simple Tes-Tris (TEST) buffer solution of 292–325 mOsm/kg as adequate extender during sperm cooling. Egg yolk, the most effective agent to protect spermatozoa against cold shock, is commonly included in semen diluents, but it is not as effective for all species; and in fact it did not increase the survival of cat spermatozoa at +5°C (Glover and Watson 1987). The protective action of egg yolk is due to the lipoproteins included in the low-density fraction (LDF). This compound protects the cell surface by interacting with the membrane (Watson 1976) and experiments conducted with cat spermatozoa demonstrated that LDF is better tolerated than egg yolk in this species (Glover and Watson 1987). The inclusion of monosaccarides (glucose, fructose and galactose) appeared to be the easiest way to provide exogenous energy substrate, but none of them offered any real advantage during storage of cat semen (Glover and Watson 1987). The cooling rate also influences spermatozoal survival (Pukazhenthi et al 1999)studied the sensitivity of felid spermatozoa to various rates of cooling temperatures and found that rapid cooling was more detrimental than slow cooling to cat spermatozoa morphological integrity. Even for epididymal spermatozoa, some authors reported a decreased motility in the semen cooled overnight compared to fresh semen (Goodrowe and Hay 1993). On the other hand, (Harris et al 2001)comparing the effect of cooling on both ejaculated and epididymal spermatozoa after 5 days of storage, reported better results of motility in epididymal semen diluted in TEST-yolk buffer compared to ejaculated semen (69 vs 51.4%), with no significant difference in viability (91 vs 88.5%). Recent work has confirmed that cat epididymal sperm cells survive after cooling, and their ability to interact with homologous oocytes is preserved. Interestingly, supplementation with antioxidants did not significantly influence motility and sperm morphology after storage (Leoni 1999), although lipid peroxidation of membranes by oxygen free radicals may be a cause of damage and loss of motility observed in cooled spermatozoa (Aitken 1995), and effects are also seen with frozen sperm (see subsequently). Semen freezingThis procedure is particularly important for endangered species, as it is a valuable tool for creating effective semen banking. It has been successfully employed both in the domestic cat with ejaculated semen (Platz et al 1976, Platz et al 1978, Pope et al 1991, Tsutsui et al 2000 and Zambelli et al 2002) and epididymal semen (Hay and Goodrowe 1993, Lengwinat and Blottner 1994 and Stachecki et al 1994), and in non-domestic felids with ejaculated semen (Howard 1986, Byers et al 1989, Donoghue et al 1992, Swanson et al 1996a and Swanson et al 1996b) and epididymal semen (Nelson et al 1999 and Bartels et al). Generally, semen can be stored in straws, ampules, or pellets (Howard 1986), although it has been shown for cat semen that the straws give better results than pellets (Pope et al 1991). Prior to freezing, semen requires an equilibration period at +5°C for about 20 min. Then it is packaged in straws or ampules and exposed to liquid nitrogen vapor by suspending 4–5 cm above the liquid nitrogen, prior to the immersion and final storage. The temperature change may also be programmed with a controlled freezer, and a suitable freezing rate has been found to be -10°C/min from +5 to -80°C, before the immersion in the liquid nitrogen (Pope et al 1991). The pelleting technique involves pipetting single drops of semen into 3-mm diameter indentations made in a block of dry ice. After a 3-min interval, the block is inverted, plunging the pellets into liquid nitrogen. The diluents for freezing cat spermatozoa are similar to those already mentioned for cooling, with a supplementation of cryoprotectant agents, with glycerol being the most widely used for this purpose. Glycerol is used at low concentration (4%) as cat spermatozoa seem to be especially sensitive to high concentration (Nelson et al 1999). It has also been reported that sperm motility in cat is more sensitive to changes in osmolality than membrane integrity, and removal of cryoprotectant in multiple steps with an isotonic solution minimizes loss of sperm motility and membrane disruption (Pukazhenthi et al 2002). However, an ideal freezing diluent for felid spermatozoa has not yet been defined, in fact sperm motility and acrosomal morphology are greatly affected by the freeze-thaw procedure. Cryopreservation of ejaculated semen was performed in the cat for the first time by (Platz et al 1976)using a diluent consisting of 20% egg yolk, 11% lactose and 4% glycerol in de-ionized water. In 1992, Donoghue et al cryopreserved tiger semen in the samemedium and reported a decline in motility of 10–40%. Similar results were obtained by (Byers et al 1989) using a TEST-yolk extender with 7.5% glycerol. (Zambelli et al 2002) demonstrated that a freezing rate of 3.85°C/min for cat semen diluted in Tris with 20% egg yolk and 4% glycerol, gave better results in spermatozoal motility and morphology preservation than faster freezing rates. Using epididymal semen, (Hay and Goodrowe 1993)compared three different extenders containing 20% egg yolk and 3% glycerol: TE (Tris buffer, citric acid and fructose), TC (Tris buffer, citric acid and glucose) and CP (lactose). In their study progressive motility of spermatozoa was reduced approximately by 20% from prefreeze values, in presence of Tris buffer and monosaccharides in the diluent TE and TC. Motility stimulants such as caffeine, pentoxifylline and 2'-deoxyadenosine had a dose-dependent effect on motility and produced a hyperactivated motion of thawed cat epididymal spermatozoa reducing the decrease in motility by approximately 10% compared to the prefreeze value (Stachecki et al 1994). As already mentioned, lipid peroxidation can induce direct membrane damage or changes in membrane structure or fluidity causing rapid and irreversible loss of motility. This event occurs in spermatozoa after cooling and even more after freezing–thawing processes. In order to face the
accumulation of reactive oxygen species during storage, the effect of
adding the antioxidant taurine (25 or 50 mM) to the diluent used for
freezing epididymal cat semen has been evaluated (Luvoni et al 2002).
Taurine, a sulfur-containing Methods of thawing frozen semen vary widely. Straws are thawed rapidly in a +35°C water bath, ampules are allowed to thaw slowly in an ice–water bath for 10 min before semen is transferred into the insemination catheter. Pellets are thawed rapidly by plunging them into a 0.9% saline solution warmed to +37°C (Howard 1986). Conception rates after AIIn spite of the reduced spermatozoal motility and morphology after thawing, frozen semen has been employed for AI purposes and the results are compared with fresh semen AI in Table 1:
The first kitten born following intravaginal insemination with frozen semen was obtained by (Platz et al 1976). Two years later, (Platz et al 1978) reported a pregnancy rate of 10% after intravaginal insemination. Such a poor result could be explained both as a consequence of the site of insemination (vagina and not uterus) and as a result of an inaccurate timing of the insemination with the expected onset of ovulation. It is well known that the site of semen deposition greatly affects the success of AI with frozen semen, because the survival time of thawed spermatozoa is significantly reduced. The development of surgical or laparoscopical intrauterine insemination, that allows sperm to be deposited close to the site of fertilization, improved the results in pregnancy rates to 57% (Tsutsui et al 2000). The application of frozen semen AI to non-domestic cats remains a problem. Attempts at intravaginal insemination of anesthetized non-domestic cats have rarely been successful (Dresser et al 1982 and Howard 1999), primarily due to compromised sperm transport and uncertainty about the ovulatory status of gonadotropin-treated females. However, laparoscopic AI has been achieved in seven non-domestic felid species. In three of them (cheetah, leopard cat and ocelot), offspring have also been produced after AI with both freshly collected and frozen-thawed spermatozoa (Howard and Doherty 1991, Howard et al 1997, Howard 1999 and Swanson et al 1996a). The development of a technique for non-surgical intrauterine insemination, that might ensure satisfactory pregnancy rates, is desirable. A practical contribution to this topic came recently from (Zambelli and Castagnetti 2001). They used a tomcat catheter with a 1 mm diameter and a nob-pointed needle inserted at the cut end to inseminate eight queens with frozen semen. Transcervical catheterization was possible in only five animals, but in all of them, embryos were recovered after ovarioisterectomy performed 5 days later. ConclusionsOne important way to prevent extinction in non-domestic felids is to establish a `frozen semen bank' and to apply ART by means of AI. This requires developing conservation protocols for semen, in order to improve reproduction that in these animals is quite poor, because of geographical isolation and population contraction that increase inbreeding and reduce genetic diversity. Moreover, the difficulties concerned with handling these animals, which make it impossible to monitor and to manipulate the female estrous cycle, contribute to the need of spreading ART. In the domestic
cat AI is indicated to optimize reproductive performances in valuable
animals or when cat is studied as a comparative model for feline
endangered species. Feline semen conservation through cooling or freezing
has so far brought some encouraging results, such as the birth of
offspring after AI with stored semen, but an optimal procedure is far from
being defined. There is a need for further investigations focused on the
diluents (such as the supplementation with cell protectants), combined
with the rate of cooling to freezing temperatures to preserve integrity
and fertilizing potential of spermatozoa.
ReferencesAitken, 1995. Free radicals, lipid peroxidation and sperm function. Reproduction, Fertility and Development 7, pp. 659–668. Bartels, P., Lubbe, K., Kilian, I., Friedman, Y., Van Dick, G. and Mortimer, D., 2000. In vitro maturation and fertilization of lion (Panthera leo) oocytes using frozen-thawed epididymal spermatozoa recovered by cauda epididymectomy of an immobilized lion (Abstr.). Theriogenology 53, p. 325. Buff, S., Donzè, A., Guerin, P., Guillaud, J., Fontbonne, A. and Menezo, Y., 2001. Taurine and hypotaurine in spermatozoa and epididymal fluid of cats. Journal of Reproduction and Fertility 57 Suppl, pp. 93–95. Byers, A.P., Hunter, A.G., Seal, U.S., Binczik, G.A., Graham, E.F., Reindl, N.J. and Tilson, R.L., 1989. In vitro induction of capacitation of fresh and frozen spermatozoa of the Siberian tiger (Panthera tigris). Journal of Reproduction and Fertility 86, pp. 599–607. CITES (1973) Convention on International Trade in Endangered Species of Wild Flora and Fauna, part of the Endangered Species Act (PL 93-205, 93rd Congress) and in 50 appendices. Code Fed. Reg., part 23. Donoghue, A.M., Johnston, L.A., Seal, U.S., Armstrong, D.L., Simmons, L.G., Gross, T., Tilson, R.L. and Wildt, D.E., 1992. Ability of thawed tiger (Panthera tigris) spermatozoa to fertilize conspecific eggs and bind and penetrate domestic cat eggs in vitro. Journal of Reproduction and Fertility 96, pp. 555–564. Dooley, M.P., Murase, K. and Pineda, M.H., 1983. An electroejaculator for the collection of semen from the domestic cat. Theriogenology 20, pp. 297–310. Dresser, B.L., Kramer, L., Reece, B. and Russell, P.T., 1982. Induction of ovulation and successful artificial insemination in a Persian leopard (Panthera pardus saxicolor). Zoo Biology 1, pp. 55–57. Glover, T.E. and Watson, P.F., 1985. The effect of buffer osmolality on the survival of cat (Felis catus) spermatozoa at 5°C (Abstr.). Theriogenology 24, p. 449. Glover, T.E. and Watson, P.F., 1987. The effects of egg yolk, the low density lipoprotein fraction of egg yolk, and three monosaccharides on the survival of cat (Felis catus) spermatozoa stored at 5°C. Animal Reproduction Science 13, pp. 229–237. Goodrowe, K.L. and Hay, M.A., 1993. Characteristics and zona binding ability of fresh and cooled domestic cat spermatozoa. Theriogenology 40, pp. 967–975. Goodrowe, K.L., Howard, J.G., Schmidt, P.M. and Wildt, D.E., 1989. Reproductive biology of the domestic cat with special reference to endocrinology, sperm function and in vitro fertilization. Journal of Reproduction and Fertility 39 Suppl, pp. 73–90. Harris, R.F., Gomez, M.C., Leibo, S.P. and Pope, C.E., 2002. In vitro development of domestic cat embryos after in vitro fertilization of oocytes with spermatozoa stored for various intervals at 4°C (Abstr.). Theriogenology 57, p. 365. Harris, R.F., Pope, C.E., Gomez, M.C., Leibo, S.P. and Dresser, B.L., 2001. Storage of domestic cat spermatozoa for extended periods at 4°C (Abstr.). Theriogenology 55, p. 308. Hay, M.A. and Goodrowe, K.L., 1993. Comparative cryopreservation and capacitation of spermatozoa from epididymides and vasa deferentia of the domestic cat. Journal of Reproduction and Fertility 47 Suppl, pp. 297–305. Herron, M.A., Barton, C.L. and Applegate, B., 1986. A modified technique for semen collection by electroejaculation in the domestic cat. Theriogenology 26, pp. 57–64. Howard JG. (1999) In: Murray ME, Fowler RC (eds), Zoo and Wild Animal Medicine. Current Therapy (4th edn). Philadelphia: WB Saunders, pp. 449–457. Howard, J.G. and Doherty, J., 1991. Leopard cats produced by artificial insemination. American Association of Zoological Parks and Aquariums Community, p. 12. Howard, J.G., Brown, J.L., Bush, M. and Wildt, D.E., 1990. Teratospermic and normospermic domestic cats: ejaculate traits, pituitary-gonadal hormones, and improvement of spermatozoal motility and morphology after swim-up processing. Journal of Andrology 11, pp. 204–215. Howard JG, Bush M, Wildt DE. (1986) In: Morrow DA (ed.), Current Therapy in Theriogenology (2nd edn). Philadelphia: WB Saunders, pp. 1047–1053. Howard, J.G., Donoghue, A.M., Barone, M.A., Goodrowe, K.L., Blumer, E.S., Snodgrass, K., Starnes, D., Tucker, M., Bush, M. and Wildt, D.E., 1992. Successful induction of ovarian activity and laparoscopic intrauterine artificial insemination in the cheetah (Acinonyx jubatus). Journal of Zoo and Wildlife Medicine 23, pp. 288–300. Howard, J.G., Roth, T.L., Byers, A.P., Swanson, W.F. and Wildt, D.E., 1997. Sensitivity to exogenous gonadotropins for ovulation induction and laparoscopic artificial insemination in the cheetah and clouded leopard (Abstr.). Journal of Andrology 55 Suppl, p. 123. Johnstone, S.D., 1984. 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Heterologous and homologous fertilizing capacity of cryopreserved felid sperm: a model for endangered species (Abstr.). Theriogenology 51, p. 290. Pineda, M.H., Dooley, M.P. and Martin, P.A., 1983. Long-term study on the effects of electroejaculation on seminal characteristics of the domestic cat. American Journal of Veterinary Research 45, pp. 1038–1041. Platz, C.C. and Seager, S.W.J., 1978. Semen collection by electroejaculation in the domestic cat. Journal of American Veterinary Medicine Association 173, pp. 1353–1355. Platz, C.C., Follis, T., Demorest, N. and Seager, S.W.J., 1976. Semen collection, freezing and insemination in the domesticcat. Proceedings of VIII International Congress on Animal Reproduction and Artificial Insemination, Cracow, pp. 1053–1056. Platz, C.C., Wildt, D.E. and Seager, S.W.J., 1978. Pregnancy in the domestic cat after artificial insemination with previously frozen spermatozoa. Journal of Reproduction and Fertility 52, pp. 279–282. Pope, C.E., Gelwicks, E.J., Wachs, K.B., Keller, G.L. and Dresser, B.L., 1989. In vitro fertilization in the domestic cat (Felis catus): a comparison between freshly collected and cooled semen (Abstr.) . Theriogenology 31, p. 241. Pope, C.E., Turner, J.L., Quatman, S.P. and Dresser, B.L., 1991. Semen storage in the domestic felid: a comparison of cryopreservation methods and storage temperature (Abstr.) . Biology of Reproduction 44 Suppl 1, p. 117. Pukazhenthi, B., Pelican, K., Wildt, D.E. and Howard, J.G., 1999. Sensitivity of domestic cat (Felis catus) sperm from normospermic versus teratospermic donors to cold-induced acrosomal damage. Biology of Reproduction 61, pp. 135–141. Pukazhenthi, B., Spindler, R., Wildt, D.E., Bush, L.M. and Howard, J.G., 2002. Osmotic properties of spermatozoa from felids producing different proportions of pleiomorphisms: influence of adding and removing cryoprotectant. Cryobiology 44, pp. 288–300. Seager SWJ. (1976) In: Klemm WR (ed.), Applied Electronics for Veterinary Medicine and Animal Physiology. Springfield: CC Thomas, pp. 410–418. Sojka, N.J. and Jennings, L.L., 1970. Collection and utilisation of cat semen for artificial insemination. Journal of American Veterinary Medicine Association 156, pp. 1250–1251. Sojka, N.J., Jennings, L.L. and Hamner, C.E., 1970. Artificial insemination in the cat (Felis catus L.). Laboratory Animal Care 20, pp. 198–204. Stachecki, J.J., Ginsburg, K.A. and Armant, D.R., 1994. Stimulationof cryopreserved epididymal spermatozoa of the domestic cat using the motility stimulants caffeine, pentoxifylline and 2'-deoxyadenosine. Journal of Andrology 15, pp. 157–164. Swanson, W.F., Howard, J.G., Roth, T.L., Brown, J.K., Alvarado, T., Burton, M., Starnes, D. and Wildt, D.E., 1996. Responsiveness of ovaries to exogenous gonadotrophins and laparoscopicartificial insemination with frozen-thawed spermatozoa in ocelots (Felis pardalis). Journal of Reproduction and Fertility 106, pp. 87–94. Swanson, W.F., Roth, T.L., Blumer, E., Citino, S.B., Kenny, D. and Wildt, D.E., 1996. Comparative cryopreservation and functionality of spermatozoa from the normospermic jaguar(Panthera onca) and teratospermic cheetah (Acinonyx jubatus) (Abstr.). Theriogenology 45, p. 241. Tanaka, A., Takagi, Y., Nakagawa, K., Fujimoto, Y., Hori, T. and Tsutsui, T., 2000. Artificial intravaginal insemination using fresh semen in cats. Journal of Veterinary Medicine Science 62, pp. 1163–1167. Tsutsui, T., Tanaka, A., Nakagawa, K., Fujimoto, Y., Murai, M., Anzai, M. and Hori, T., 2000. Unilateral intrauterine horn insemination of frozen semen in cats. Journal of Veterinary Medicine Science 62, pp. 1247–1251. Watson, P.F., 1976. The protection of ram and bull spermatozoa by the low density lipoprotein fraction of egg yolk during storage at 5°C and deep-freezing. Journal of Thermal Biology 1, pp. 137–141. Wildt DE. (1990) In: Bavister BD, Cummins J, Roldan ERS (eds), Fertilization in Mammals. Norwell: Serono Symposium, pp. 349–364. Zambelli, D. and Belluzzi, S., 1998. Raccolta e valutazione del materiale seminale di gatto. Praxis Veterinaria 1, pp. 20–22. Zambelli, D. and Castagnetti, C., 2001. Transcervical insemination with fresh or frozen semen in the domestic cat: new technique and preliminary results (Abstr.). European Society of Domestic Animal Reproduction Newsletter 6, p. 34. Zambelli, D., Caneppele,
B., Castagnetti, C. and Belluzzi, S., 2002. Cryopreservation of cat semen
in straws: comparison of five different freezing rates. Reproduction in
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Feline semen characteristicsOnce semen is obtained, and before its use for assisted reproduction techniques (ART), the evaluation of sperm quality is a necessary step in order to know whether the sample of semen is suitable for conservation treatment. It is important to note that cats, as well as non-domestic felids, are very often affected by teratospermia, a condition in which more than 60% of the spermatozoa show aberrant forms (in normal laboratory cats this is less than 30%) (Pukazhenthi et al 2001). Predominant anomalies include a bent mid-piece with or without a cytoplasmic droplet, a bent flagellum and a tightly coiled flagellum. Moreover, high proportions of spermatozoa show acrosomal defects, such as large vacuoles, protrusion of acrosomal matrix and folding of the acrosome back on to itself (Pukazhenthi et al 2001). The etiology of teratospermia in the domestic cat is unknown, but structurally defective spermatozoa observed in wild felids, such as the cheetah (Wildt et al., 1987a; Wildt et al., 1984; Wildt et al., 1987b and Wildt et al., 1988) and geographically isolated lion populations ( Wildtet al 1987a) have been related to decreased genetic variation and low circulating testosterone concentrations. Howard et al (1990) demonstrated that testosterone concentrations in teratospermic cats are 33% lower than in normospermic males. Motility of ejaculated spermatozoa can be affected by morphological anomalies. Percent motility and association of morphological anomalies in domestic cat and wild felids have been reported and are described in Table 1:
Experiments conducted using cat epididymal spermatozoa show that they are more frequently affected by morphological abnormalities, with values ranging from 36 to 54% (Goodrowe and Hay, 1993; Hay and Goodrowe, 1993 and Lengwinat and Blottner, 1994). This is partially due to the fact that spermatozoa continue the maturation process in epididymides and vasa deferentia, and samples obtained from these structures often contain immature forms. Motility of epididymal sperm is reported to be 71–79% (Goodrowe and Hay, 1993; Hay and Goodrowe, 1993; Stachecki et al., 1993; Lengwinat and Blottner, 1994 and Stachecki et al., 1994) and 57% ( Stachecki et al 1993) in normospermic andteratospermic cats, respectively. Cold-induced damageIn felids, fertility from artificial insemination (AI) with stored semen is poorer than fertility with fresh semen, a fact that can only be partially compensated for by inseminating greater numbers of live spermatozoa close to the site of fertilization by means of an intrauterine insemination (Tsutsui et al 2000). The cryopreservation process includes several steps, from sperm preparation and dilution to the post-thawing maintenance of functional capability: at each of these steps, spermatozoa can lose their ability to function normally. As a result of cooling, some workers have reported a decreased motility in ejaculatedspermatozoa compared with fresh semen (43.5 vs 52.2%; Glover and Watson 1985), decreased progressive motility and altered morphology with evidence of acrosomal damage. Pukazhenthi et al (1999)reported a marked decline in intact acrosomes (65.6 vs 81.5% in the fresh sample). Motility and acrosomal integrity are also severely affected by the freezing process. In fact, Wood et al (1993)reported for ejaculated cat sperm a marked decrease in intact acrosomes (28 vs 90% in the fresh sample), and similar results were obtained by Swanson et al (1996b)in the jaguar andcheetah. Acrosomal damage has been reported even in epididymal frozen–thawed semen. Hay and Goodrowe (1993)observed 30–50% loss of acrosomal integrity. Lengwinat and Blottner (1994) reported a decreased motility (75.8 vs 53%) and decreased acrosomal integrity (69.5 vs 22.5%) after thawing. Therefore, cooled andcryopreserved sperms undergo severe damages that alter their motility and morphology, thus affecting their fertilizing potential. This is explained by the fact that some stages of the process can be very stressful, such as the change in temperature (cold shock), as well as the formation and dissolution of ice crystals. The mechanism by which cold shock acts on the sperm cell is not entirely clear, but it is probably related to the phase transitions of membrane lipids, resulting in phase separations and loss of the selective permeability characteristics of living biological membranes (Watson 1995). Ice crystals cause rupture of cellular membranes when the cells are exposed to a rapid rate of cooling (4°C/min) from body temperature to +5°C, while decreasing the cooling rate to 0.5°C/min minimizes the structural damage (Pukazhenthi et al 1999). As the temperature is reduced below 0°C, the water content in the extracellular medium undergoes crystallization. The solute is concentrated in the remaining fluid portion, and the cell is subjected to osmotic dehydration and shrinkage due to efflux of intracellular water (Watson 1995). The cryoprotectant agent usually present in the freezing diluent (glycerol) provides protection to the cells from the consequences of ice formation by increasing the unfrozen water fraction, but the osmotic effect of molar concentrations of this compound may result in membrane damage. Moreover, the toxicity of glycerol for cat spermatozoa has been reported (Nelson et al 1999). The decline in motility could be due to changes in osmolality (Pukazhenthi et al 2002) and in active transport and permeability of the plasma membrane in the tail region, combined with an alteration of energy availability or damage to theaxonema elements (Watson 1995). In the cell, several of the organelles are enveloped by a membrane, which is particularly vulnerable during the cryopreservation cycle, and many of the cytoskeleton proteins exhibit a temperature-dependent depolymerization and repolymerization, which could have severe implications for sperm cell viability (Watson 1995). Thus, frozen–thawed spermatozoa demonstrate a loss of internal mitochondrial structure (Watson 1979). Even the nucleus can be altered during cryopreservation, and the degree of denaturation of DNA is influenced by diluents (Karabinus et al 1991). It is important that spermatozoa from teratospermic males are more susceptible to cold and osmotic stress, which induce membrane disruption, than those from normospermic males (Pukazhenthi et al 1999). These observationsindicate that there may be membrane differences between spermatozoa from normospermic vs teratospermic donors that, in turn, may influence the kinetics of water and solute movement across membranes. One of the consequences of destabilization of membranes by cooling or cryopreservation is a premature acrosome reaction that shortens the life span of the spermatozoa and reduces fertility. In fact, cryopreserved spermatozoa may be considered to be in a state resembling partially capacitated spermatozoa. Their membranes have undergone similar fluidity changes as those seen during capacitation; they are permeable to calcium ions that promote both capacitation and the acrosome reaction. Their viability is thus limited because capacitated spermatozoa do not have a prolonged survival: they are usually activated close to the time of meeting the oocyte. Moreover, a false acrosome reaction has also been described in thawed spermatozoa, in which acrosomal changes are associated with irreversible membrane damages (Bedford, 1970 and Meizel, 1978). It has been reported that a significant higher proportion of cat epididymal spermatozoa after thawing showed the pattern of acrosome reaction compared with fresh or diluted semen before freezing. The proportion of acrosome-reacted spermatozoa in frozen–thawed samples indicates that many cells were damaged during freezing and thawing and, therefore, are functionally compromised (Marinoni 2001). Spermatozoa must undergocapacitation and the acrosomal reaction in order to penetrate the oocyte, and this is possible only if sperm acrosomal integrity has not been altered during storage. Assessment of motility and morphologyMotility may be evaluated as percent motility and progressive motility, and the latter is classified according to a scale ranging from 0 (no movement) to 5 (steady, rapid forward progression). From these two parameters, a spermatozoamotility index (SMI) can be derived:SMI=0.5×[(progressive motility×20)+(%) motility] (Howard 1993). Another method for determining motion characteristics of cat spermatozoa is Computer Assisted Semen Analysis (CASA), which makes it possible to measure different kinematic parameters, such as curvilinearvelocity, linearity, straight-line velocity and amplitude of lateral head displacement. The CASA technique can evaluate various motility parameters, and it could be useful for identifying a hyperactivated status in spermatozoa, which could suggest their capacitated condition (Stachecki et al 1993). Morphology of cat spermatozoa can be evaluated fixing a semen sample in glutaraldehyde 1% and evaluating spermatozoa with a phase contrast microscopy, or with all the usual stains used in semen analysis, like eosin–nigrosin, eosin–fast green FCF etc (Byers et al., 1989 and Hay and Goodrowe, 1993). A staining technique (1% rose bengal, 1% fast green FCF and 40% ethanol in citric acid–disodium phosphate buffer) for evaluating acrosomal morphology of feline spermatozoa was described by Pope et al (1991). There are several classifications of spermatozoal morphological abnormalities in fresh semen, and the one most commonly used differentiates them into: primary, when they occur during spermatogenesis in the testicles, such as a coiled flagellum, and microcephalic or macrocephalic defect and secondary, due to damages during their maturation and transport along the epididymes, such as a bent mid-piece, a bent flagellum, protoplasmic droplets and acrosomal anomalies. Primary defects are normally considered more detrimental to fertility than secondary deformities (Wildt et al., 1987a and Howard et al., 1990). With regard to preserved semen, the evaluation of abnormalities focuses on the acrosome, since this structure is vulnerable and often altered by cooling and freezing processes. Accurate methods for evaluation of cold-induced damage on acrosome membrane as indicator of fertilization ability have been developed and are described subsequently. Assessment of spermatozoal fertilizing abilityMethods of evaluating capacitation status and acrosomal reaction of cat spermatozoa can be classified into biological assays and fluorescent staining techniques. Biological assaysInclude different types of in vitro assays, such as:
Fluorescent staining techniquesInclude:
ConclusionsThe final target of semen conservation is the preservation of spermatozoal fertilizing potential in order to develop ART and to increase felid survival chances. Studies conducted on conservation protocols have produced important results in domestic cat as well as in wild felids, but have also elicited many problems, most of which are still unsolved. Cooled and cryopreserved sperms undergo several types of damage, which alter their motility and morphology and are responsible for low pregnancy rate after AI. For these reasons, there
is the need to define the ideal diluent components and to optimize cooling
and freezing protocols to prevent membrane modification. Moreover, it is
necessary to study these damages and to develop new methods to evaluate
them. Among currently applied methods, biological assays and fluorescent
staining techniques not only allow a morphological evaluation of the
spermatozoa, but also provide information on its functional integrity. The
CTC, in particular, allows monitoring of various transitional phases that
spermatozoa display from capacitation to acrosome reaction resulting from
cold-induced damages.
ReferencesAndrews, J.C., Howard, J.G., Bavister, B.D. and Wildt, D.E., 1992. Sperm capacitation in the domestic cat (Felis catus) and leopard cat (Felis bengalensis) as studied with a salt-stored zonapellucida penetration assay. Molecular Reproduction and Development 31, pp. 200–207. Bartels, P., Lubbe, K., Kilian, I., Friedman, Y., Van Dick, G. and Mortimer, D., 2000. In vitro maturation and fertilization of lion (Panthera leo) oocytes using frozen–thawed epididymal spermatozoa recovered by cauda epididymectomy of an immobilized lion (abstract) . Theriogenology 53, p. 325. Bedford, J.M., 1970. Sperm capacitation and fertilization in mammals. Biology of Reproduction 2 Suppl, pp. 128–158. Brown, J.L., Wildt, D.E., Phillips, L.G., Seidensticker, J., Fernando, S.B.U., Miththapala, S. and Goodrowe, K.L., 1989. Adrenal–pituitary–gonadal relationship and ejaculate characteristics in captive leopards (Panthera pardus kotiya) isolated on the island of Sri Lanka. Journal of Reproduction and Fertility 85, pp. 605–613. Byers, A.P., Hunter, A.G., Seal, U.S., Binczik, G.A., Graham, E.F., Reindl, N.J. and Tilson, R.L., 1989. In-vitro induction of capacitation of fresh and frozen spermatozoa of the Siberian tiger (Panthera tigris). Journal of Reproduction and Fertility 86, pp. 599–607. Da Paz, R.C.R., Züge, R.M., Morato, R.G., Barnabe, R.C. and Barnabe, V.H., 2002. Penetration assay of frozen jaguar (Panthera onca) sperm in heterologous oocytes (abstract) . Theriogenology 57, p. 588. Donoghue, A.M., Johnston, L.A., Seal, U.S., Armstrong, D.L., Simmons, L.G., Gross, T., Tilson, R.L. and Wildt, D.E., 1992. Ability of thawed tiger (Panthera tigris) spermatozoa to fertilize conspecific eggs and bind and penetrate domestic cat eggs in vitro. Journal of Reproduction and Fertility 96, pp. 555–564. Glover, T.E. and Watson, P.F., 1985. The effect of buffer osmolality on the survival of cat (Felis catus) spermatozoa at 5°C (abstract) . Theriogenology 24, p. 449. Goodrowe, K.L. and Hay, M.A., 1993. Characteristics and zona binding ability of fresh and cooled domestic cat spermatozoa. Theriogenology 40, pp. 967–975. Hay, M.A. and Goodrowe, K.L., 1993. Comparative cryopreservation and capacitation of spermatozoa from epididymides and vasa deferentia of the domestic cat. Journal of Reproduction and Fertility 47 Suppl, pp. 297–305. Howard JG (1993) In: Zoo and Wild Animal Medicine. Current Therapy (3rd edn). Fowler RC (ed), Philadelphia: WB Saunders, pp. 390–399. Howard, J.G. and Wildt, D.E., 1990. Ejaculate-hormonal traits in the leopard cat (Felis bengalensis) and sperm function as measured by in vitro penetration of zona-free hamster and zona intact domestic cat oocytes. Molecular Reproduction and Development 26, pp. 163–174. Howard, J.G., Brown, J.L., Bush, M. and Wildt, D.E., 1990. Teratospermic and normospermic domestic cats: ejaculate traits, pituitary–gonadal hormones, and improvement of spermatozoal motility and morphology after swim-up processing. Journal of Andrology 11, pp. 204–215. Howard JG, Bush M, Wildt DE (1986) In: Current Therapy in Theriogenology (2nd edn). Morrow DA (ed), Philadelphia: WB Saunders, pp. 1047–1053. Karabinus, D.S., Evenson, D.P. and Kaproth, M.T., 1991. Effects of egg yolk-citrate and milk extenders on chromatin structure and viability of cryopreserved bull sperm. Journal of Dairy Science 74, pp. 3836–3848. Lengwinat, T. and Blottner, S., 1994. In vitro fertilization of follicular oocytes of domestic cat using fresh and cryopreserved epididymal spermatozoa. Animal Reproduction Science 35, pp. 291–301. Leoni S (1999) Refrigerazione del seme epididimale felino in diversi diluenti. Tesi di Laurea, Facoltà di Medicina Veterinaria, Università degli Studi di Milano. Marinoni G (2001) Impiego della clortetraciclina nella valutazione dell'integrità funzionale degli spermatozoi epididimali felini congelati. Tesi di Laurea, Facoltà di Medicina Veterinaria, Università degli Studi di Milano. Meizel (1978) In: Development in Mammals Vol. 3. Johnson M (ed), Amsterdam: Elsevier/North Holland Biomedical Press, pp. 1–64. Nelson, K.L., Crichton, E.G., Doty, L., Volenec, D.E., Morato, R.G., Pope, C.E., Dresser, B.L., Brown, C.S., Armstrong, D.L. and Loskutoff, N.M., 1999. Heterologous and homologous fertilizing capacity of cryopreserved felid sperm: a model for endangered species (abstract) . Theriogenology 51, p. 290. Pope, C.E., Gelwicks, E.J., Wachs, K.B., Keller, G.L. and Dresser, B.L., 1989. In vitro fertilization in the domestic cat (Felis catus): a comparison between freshly collected and cooled semen (abstract) . Theriogenology 31, p. 241. Pope, C.E., Zhang, Y.Z. and Dresser, B.L., 1991. A simple staining method for evaluating acrosomal status of cat spermatozoa. Journal of Zoo and Wildlife Medicine 22, pp. 87–95. Pukazhenthi, B.S., Noiles, E., Pelican, K., Donoghue, A., Wildt, D.E. and Howard, J.G., 2000. Osmotic effects on feline spermatozoa from normospermic versus teratospermic donors. Cryobiology 40, pp. 139–150. Pukazhenthi, B., Pelican, K., Wildt, D.E. and Howard, J.G., 1999. Sensitivity of domestic cat (Felis catus) sperm from normospermic versus teratospermic donors to cold-induced acrosomal damage. Biology of Reproduction 61, pp. 135–141. Pukazhenthi, B., Spindler, R., Wildt, D.E., Bush, L.M. and Howard, J.G., 2002. Osmotic properties of spermatozoa from felids producing different proportions of pleiomorphism: influence of adding and removing cryoprotectant. Cryobiology 44, pp. 288–300. Pukazhenthi, B., Wildt, D.E. and Howard, J.G., 2001. The phenomenon and significance of teratospermia in felids. Journal of Reproduction and Fertility 57 Suppl, pp. 423–433. Roth, T.L., Howard, J.G., Donoghue, A.M., Swanson, W.F. and Wildt, D.E., 1994. Function and culture requirements of snow leopard (Panthera uncia) spermatozoa in vitro. Journal of Reproduction and Fertility 101, pp. 563–569. Saling, P.M. and Storey, B.T., 1979. Mouse gamete interactions during fertilization in vitro. Chlortetracycline as a fluorescent probe for the mouse sperm acrosome reaction. Journal of Cell Biology 83, pp. 544–555. Stachecki, J.J., Ginsburg, K.A. and Armant, D.R., 1994. Stimulation of cryopreserved epididymal spermatozoa of the domestic cat using the motility stimulants caffeine, pentoxifylline and 2'-deoxyadenosine. Journal of Andrology 15, pp. 157–164. Stachecki, J.J., Ginsburg, K.A., Leach, R.E. and Armandt, D.R., 1993. Computer-assisted semen analysis (CASA) of epididymal sperm from the domestic cat. Journal of Andrology 14, pp. 60–65. Swanson, W.F., Howard, J.G., Roth, T.L., Brown, J.K., Alvarado, T., Burton, M., Starnes, D. and Wildt, D.E., 1996. Responsiveness of ovaries to exogenous gonadotrophins and laparoscopicartificial insemination with frozen–thawed spermatozoa in ocelots (Felis pardalis). Journal of Reproduction and Fertility 106, pp. 87–94. Swanson, W.F., Roth, T.L., Blumer, E., Citino, S.B., Kenny, D. and Wildt, D.E., 1996. Comparative cryopreservation and functionality of spermatozoa from the normospermic jaguar (Panthera onca) and teratospermic cheetah (Acinonyx jubatus) (abstract) . Theriogenology 45, p. 241. Tsien RY (1989) In: Fluorescence Microscopy of Living Cells in Culture. Part B. Quantitative Fluorescence Microscopy––Imaging and Spectroscopy Vol. 30. Taylor, DL, Wang YL (eds), New York: Academic Press, pp. 127–156. Tsutsui, T., Tanaka, A., Nakagawa, K., Fujimoto, Y., Murai, M., Anzai, M. and Hori, T., 2000. Unilateral intrauterine horn insemination of frozen semen in cats. Journal of Veterinary Medicine Science 62, pp. 1247–1251. Watson PF (1979) The preservation of semen in mammals. In: Oxford Reviews of Reproductive Biology Vol. 1. Finn CA (ed), Oxford: Oxford University Press, pp. 283–351. Watson, P.F., 1995. Recent developments and concepts in the cryopreservation of spermatozoa and the assessment of their post-thawing function. Reproduction, Fertility and Development 7, pp. 871–891. Wildt, D.E., Bush, M., Goodrowe, K.L., Packer, C., Pusey, A.E., Brown, J.L., Joslin, P. and O'Brien, S.J., 1987. Reproductive and genetic consequences of founding isolated lion populations. Nature 329, pp. 328–331. Wildt, D.E., Bush, M., Howard, J.G., O'Brien, S.J., Meltzer, D., Van Dick, A., Ebedes, H. and Brand, D.J., 1983. Unique seminal quality in the south African cheetah and a comparative evaluation in the domestic cat. Biology of Reproduction 29, pp. 1019–1025. Wildt, D.E., Melzer, D., Chakraborty, P.M. and Bush, M., 1984. Adrenal–testicular–pituitary relationship in the cheetah subjected to anesthesia/electroejaculation. Biology of Reproduction 30, pp. 665–672. Wildt, D.E., O'Brien, S.J., Howard, J.G., Caro, T.M., Roelke, M.E., Brown, J.L. and Bush, M., 1987. Similarity in ejaculate-endocrine characteristics in captive versus free-ranging cheetahs of two subspecies. Biology of Reproduction 36, pp. 351–360. Wildt, D.E., Phillips, L.G., Simmons, L.G., Chakraborty, P.K., Brown, J.L., Howard, J.G., Teare, A. and Bush, M., 1988. A comparative analysis of ejaculate and hormonal characteristics of the captive male cheetah, tiger, leopard and puma. Biology of Reproduction 38, pp. 245–255. Wood, T.C., Swanson,
W.F., Davis, R.M., Anderson, J.E. and Wildt, D.E., 1993. Functionality of
sperm from normo- versus teratospermic domestic cats cryopreserved in
pellets or straw containers (abstract). Theriogenology 39,
p. 342. Copyright © 2003 ESFM and AAFP. Published by Elsevier Science Ltd. (Journal of Feline Medicine & Surgery).
. The York Chocolate could be considered genetically a very particular cat breed. Few phenotypic peculiarities of the York are object of scientific studies and very interesting researches in genetic applications and not only. Preservation, diffusion and universal recognition of the York Chocolate in the world must be considered a mission not only because of the intrinsic beauty but especially because its genetic particularities allows to many experts to develop new discoveries and genetic theories. The phenotypic peculiarities characterizing the breed may be considered perfectly fixed and transferable constantly on all descendants. These peculiarities may be listed as follows: color hair, white displacement on the coat and under-coat’s lack. Today, every world feline lovers and genetic experts are very interesting on the last two features of the York.
The lacking under-coat Lacking under-coat surely is the most important phenotype characterizing the breed. Many researches and crossing experiments would confirm the existence of a gene controlling the under coat expression. In particular, collecting many informations from York Chocolate pedigree and applying statistical formulations on a lot of observations the Mendel lows would result perfectly verified. Today, we look on new observations and crossing experiments in order to have soon the absolute confidence of the gene individuation.
The GENETIC HYPOTHESIS formulation may be explained as follows: The
gene controlling under coat expression has two alleles: The
wild allele “yuc” recessive
producing a normal thick under coat. The initials of the following words York chocolate, under coat obtain the Yuc acronym. Veterinary Department of Pisa University is very interested about the individuation of this gene, because it’s common opinion to consider cat and dog under coat the principal vehicle of many human allergies. The existence of a gene controlling these phenotypes could revolutionize all the classical research approaches in order definitively to solve this very common pathology. Another interesting aspect about the under coat gene identification is the possibility to transfer on other breed this hypoallergenic feature.
Another interesting York Chocolate physic expression surly is the particular white displacement on the coat of the bicolor subjects. Janet Chiefari, the first York breeder and Anna Baldi first European breeder just fixed a particular white displacement on the coat maintaining the same configuration in all successive generations. The white displacement on the York’s coat is called Spotting Particolor. In literature, several genetic experts kept an open mind about the existence of a allele called “particolor” inside the White Spotting gene. The White spotting gene control presence and pattern of white masking the normal coat pattern, and has four alleles: non-spotted, "s", spotted, "S", particolor, "Sp", and Birman, "sb".
* The spotted allele, “S”, is mutant, is dominant, and produces white spotting which masks the true coat color in the affected area. This is a variably-expressed allele with a very wide expression range: From a black cat with one white hair to a white cat with one black hair.
*
The Birman allele, “Sb”,
if it exists, is a variation of the spotted allele producing white feet.
Variable expression ranges from white legs and feet to white toes only. * Finally the particolor allele, “Sp”, if it exists, is a variation of the spotted allele affecting the pattern of white. The classic particolor pattern is an inverted white "V" starting in the center of the forehead and passing through the centers of the eyes. The chin, chest, belly, legs and feet are white. Variable expressions of this allele range downward to a simple white locket or a white spot on the forehead. The York chocolate breed would totally confirm the Particolor allele existence. Observing the “Sp” frequency expression of white pattern transmission on several bicolor York chocolate of the Janet Chiefari and Anna Baldi breeders would confirm perfectly the inherit factors of the Mendel lows. On
conclusion, York chocolate is the exclusive breed fixing the particolor
white displacement on the bicolor subject as well as the Birman is the
only cat breed having the typical white gloves. Stability
about York chocolate phenotypes
Genes and polygenes transmission controlling the singular phenotypes of the York Chocolate breed may be considered perfectly stabilized. To day, a perfect and original standard describing exhaustively the body conformation and a complete genetic map consisting of all possible allele combinations of the York has been performed. This very meticulous genetic work about classification of all York chocolate phenotypic expressions had two advantages:
*
To have a detailed scientific description of the breed . Gene
and Allele classification correlated with the York chocolate physic
expressions A
few of the common cat genes have been identified and mapped. These genes
are grouped according to the effects they have: the body-conformation
genes which affect the shape of the body parts; the coat-conformation
genes which affect the texture and length of the coat; and the
color-conformation genes which affect the color and pattern of the coat. The
color-conformations genes are themselves divided into three groups: the
color genes which control the color of the coat and its density; the
color-pattern genes which control the pattern of the coat and expression
of the color; and the color-masking genes which control the degree and
type of masking of the basic color. On
the next cat gene list, an asterisk closed the gene acronym will identify
its existence inside the York DNA and a synthetic description will be
reported. .The
Body-Conformation Genes
The
Scottish-fold gene .The
Coat-Conformation Genes
The
Sphinx gene:
the Longhaired gene à
short
or long coat. The wild allele, "L", is dominant and produces a
normal shorthaired coat. The mutation, "l", is recessive and
produces the longhaired coat of the Persians, Angoras, Main Coons, and
others. The
Cornish Rex gene The York under coat gene à: The wild allele “yuc” is recessive and produces a normal thick under coat The
mutation allele “Yuc” is dominant and produces a total under coat lack The Color-Conformation Genes* The Color Gene à this gene controls the actual color of the coat and comes in three alleles: 1)The black allele, "B", is wild, is dominant, and produces a black or black-and-brown tabby coat. 2)The dark-brown allele, "b", is mutant, is recessive to black but dominant to light brown, and reduces black to dark brown. 3)The
light-brown allele, "bl", is mutant, is recessive to both black
and dark brown, and reduces black to a medium brown. *
The Orange-Making Gene *
The Color-Density Gene à
controlling the
coat color is the color-density gene. This gene controls the uniformity of
distribution of pigment throughout the hair and comes in two alleles:
1)dense, "D", and 2)dilute, "d". * The Albinism Gene à controls the amount of body color and comes in five alleles: 1)The full color allele, "C" is wild, is dominant, and produces a full expression of the coat colors. This is sometimes called the non- albino allele. 2)The Burmese allele, "cb", is mutant, is recessive to the full color allele and produces a slight albinism, reducing black to a very dark brown, called sable in the Burmese breed, and producing green or green-gold eyes. 3)The Siamese allele, "cs", is mutant produces an intermediate albinism, reducing the basic coat color from black/brown to a light beige with dark brown "points" in the classic Siamese pattern and producing bright blue eyes. 4)The blue-eyed albino allele, "ca", is mutant, is recessive to the full color, Burmese and Siamese alleles and dominant to the albino allele, and produces a nearly complete albinism with a translucent white coat and very washed-out pale blue eyes. 5)The
albino allele, "c", is mutant, is recessive to all others, and
produces a complete albinism with a translucent white coat and pink eyes. * The Agouti Gene à controlling the pattern of the coat and comes in two alleles: 1) The agouti allele, "A", is wild, is dominant, and produces a banded or ticked (agouti) hair, which in turn will produce a tabby coat pattern. 2)
The non-agouti allele, "a", is mutant, is recessive, and
suppresses ticking, which in turn will produce a solid-color coat. *
The
Tabby Genes * The Color-Inhibitor Gene à controls the expression of color within the hair and comes in two alleles: 1) The non-inhibitor allele, "i", is wild, is recessive, and allows expression of the color throughout the length of the hair, producing a normally colored coat. 2)
The inhibitor allele, "I", is mutant, is dominant, and inhibits
expression of the color over a portion of the hair. * The Spotting Gene à color expression is the white-spotting gene and has four alleles: 1) The non-spotted allele, "s", is wild, is recessive, and produces a normal coat without white. 2) The spotted allele, "S", is mutant, is dominant, and produces white spotting which masks the true coat color in the affected area. 3)The particolor allele, "Sp", if it exists, is a variation of the spotted allele affecting the pattern of white. 4)The
Birman allele, "Sb", if it exists, is a variation of the spotted
allele producing white feet. * The Dominant-White Gene à expression of white coat: 1) the white allele ”W” is dominant and produces white coat with blue or orange eyes. 2) the recessive allele ”w” produces the total expression of color.
Genetic Table reporting all allele combinations about all the possible York chocolate physical expressions:The columns (composed by two cells) classify the genes expressing the physical conformations. On the first row we have alleles controlling the phenotypic expressions. Second row showing recessive alleles.
Self Lavander (York Chocolate):
Self Chocolate (York Chocolate): PHENOTYPE l Yuc b D C a i s w RECESSIVE l Yuc b d C a i s w .
PHENOTYPE l Yuc b D C a i s w RECESSIVE l Yuc b D C a i s w
Lavander bicolor Particolor (York Chocolate): PHENOTYPE l Yuc b d C a i Sp w RECESSIVE l Yuc b d C a i s w
.
PHENOTYPE l Yuc b d C a i Sp w RECESSIVE l Yuc b d C a i Sp w Chocolate bicolor Particolor (York Chocolate):
.
.
PHENOTYPE l Yuc b D C a i Sp w RECESSIVE l Yuc b d C a i Sp w .
PHENOTYPE l Yuc b D C a i Sp w RECESSIVE l Yuc b D C a i Sp w .
. References Fulvio
Bresciani was born in Saluzzo (Italy) on December 12th, 1960.
He conferred a degree in Nuclear Engineering on February 1985. Since 1987
working in optical design and signal processing applied on spatial
technologies. He is author of many technical papers and several
international publications. Last
time studying new mathematical models based on quantitative genetic
principles in order to solve multivariable problems of non-linear systems.
Together with Pisa University developing mathematical models predicting
hereditary factors on feline selection. The more recently research
developed regards the individuation of a new gene controlling the under
coat appearance. Bibliografy [1]
Feline Genetics Autori: R.
Roger Breton, Nancy J Creek – Edito (netpets 199 ********** .
Lo York Chocolate e` geneticamente una razza molto particolare, tanto che, alcune sue peculiarità`, sono oggetto di studi scientifici che potrebbero portare ad interessanti sviluppi nell’individuazione di nuovi geni, con ricadute su tutto il mondo felino e non solo. La salvaguardia, la diffusione e l’universale riconoscimento della razza York Chocolate non sono importanti esclusivamente per l’intrinseca bellezza e particolarità` di questo gatto, ma anche perché stanno permettendo e permetteranno a scienziati ed appassionati di poter continuare a studiare quelle particolarità` genetiche di questa razza, essendo unica portatrice di quelle espressioni fenotipiche. Le espressioni fisiche ormai assolutamente stabilizzate che tutte insieme caratterizzano la razza, si possono elencare come segue: Il colore del pelo, la disposizione del bianco nella versione bicolore e la mancanza totale di sottopelo. In
particolare, due di queste espressioni fisiche, ovvero, la mancanza di sottopelo
e la particolare conformazione della macchia bianca che quando e` presente e`
costantemente sempre uguale, hanno e stanno interessando i genetisti ed
appassionati del mondo felino e non solo. La
mancanza di sottopelo La
mancanza di sottopelo, e` geneticamente una peculiarità che sta portando,
forse, alla scoperta genetica più
interessante associata a questa razza, ovvero l’individuazione di un nuovo
gene che regola per l’appunto l’espressione del sottopelo. La costante
ereditabilita` di quest’espressione fisica in quasi tutti i soggetti
selezionati, mi ha particolarmente attratto, facendomi supporre che ciò potesse
essere regolato da un particolare gene. Ulteriori
osservazioni fatte su incroci mirati per verificare che le frequenze attese dei
fenotipi associati al presunto gene, verificassero le frequenze delle leggi di
Mendel hanno avuto successo. Ancora
alcune osservazioni che, se andassero come presumo a buon fine, darebbero un
grado di confidenza altissimo dell’esistenza del gene. L’IPOTESI GENETICA su cui sto lavorando e` la seguente: Gene
che regola l’espressione del sottopelo si presenta con due versioni di alleli yuc “allele
selvatico” e recessivo comanda l’espressione della presenza del sottopelo Y sta per York
Chocolate la razza attraverso lo studio della quale mi ha permesso
l’individuazione del gene A questo
studio sull’individuazione del gene, e` particolarmente interessata
l’Università` di Pisa in particolare “l’Istituto di Genetica degli
Animali da Compagnia” della Facoltà` di Veterinaria.. Questo interesse,
deriva, oltre che per la scoperta in se, anche perché e` comune opinione (in
via di accertamento scientifico), che molte allergie negli uomini, dovute alla
presenza di gatti, non dipendano esclusivamente dalla saliva ma anche dal
sottopelo. La possibilità quindi di poter geneticamente controllare
l’espressione fisica della mancanza di sottopelo, permetterebbe la selezione,
indipendentemente dalla razza, di individui felini ipoallergici. La
disposizione del bianco negli individui bicolore Un’altra
caratteristica molto particolare di questa razza sicuramente originale, e` la
particolare conformazione del bianco nei soggetti bicolore. Sia Janet Chiefari,
la prima selezionatrice degli York Chocolate, che Anna Baldi, la principale allevatrice di York
in Europa, sono riusciti a stabilizzare definitivamente la conformazione
geometrica del bianco negli individui bicolore secondo quella disposizione che
geneticamente viene definita Spotting Particolor. Da molti
anni i genetisti si interrogano sul fatto se esista l’allele chiamato
Particolor (rappresentato in forma compatta come “Sp”) come variazione
dell’allele “Spotting” del gene White Spotting. Il
gene White
Spotting,
e` il gene che regola l’espressione del bianco in forma di macchie sul
mantello del gatto. Il bianco a
macchie, se e` presente sul mantello, copre tutti gli altri colori.
Ora, secondo una teoria, la cui selezione dello York la confermerebbe
almeno in parte, Il gene White
spotting esisterebbe sotto forma di
4 alleli: * “S” dominante e mutante e` associato alla presenza di bianco sul mantello. Il bianco, in questo caso, copre tutti i colori e può presentarsi secondo una infinita gamma di conformazioni , dimensioni e disposizioni geometriche che vanno da un gatto nero con un solo pelo bianco a un gatto bianco con un solo pelo nero. * “s” recessivo e “di tipo selvatico” e` associato alla totale mancanza di bianco sul mantello. * “Sb” allele birmano, se esiste e` una variazione dell’allele Spotting, ed e` associato alle presenza del bianco sulle zampe. Esso può variare dal coprire completamente le zampe fino a soltanto ad essere confinato sulle estremità delle dita. L’allele prende il nome dal gatto la cui caratteristica e` un elemento di distinzione della razza, il Sacro di Birmania.
* “Sp”,
allele Particolor, se esiste e` una variazione dell’allele Spotting e
si presenta in quella classica conformazione geometrica che
caratterizza lo York Chocolate
bicolore. Il motivo Particolor classico e`, la presenza di bianco che,
partendo dal naso come una V rovesciata, continua coprendo tutto il mento, il
collo, il petto, e tutte o solo parte delle zampe. Una variante espressione di
questo allele, e` la presenza di una sola macchia bianca più` o meno grande, al
centro del petto.
La razza York Chocolate, confermerebbe pienamente questa teoria. Un’analisi di genetica quantitativa sulla severa selezione di questa particolare disposizione del bianco sul mantello iniziata da Janet Chiefari, per arrivare all’allevatrice Anna Baldi dimostrerebbero esattamente questa teoria. Le frequenze di espressione dell’allele “Sp” ottenute dai vari incroci tra soggetti di tipo Particolor e colorazione Solida, hanno statisticamente confermato le frequenze teoriche attese dalle leggi di Mendel. Concludendo
si può affermare che, l’isolamento di questa variazione dell’allele
Spotting, e` una peculiarita` esclusiva della razza York
Chocolate allo stesso modo dell’allele birmano che caratterizza la razza
del Sacro di Birmania. Stabilizzazione
della razza York Chocolate Lo
York Chocolate e` una razza le cui espressioni fisiche, sia quelle controllate
dai singoli geni che dai poligeni, sono perfettamente stabilizzate e si ripetono
costantemente con variazioni minime in tutti i discendenti. Lo standard e`
perfettamente delineato, in particolare e` stata completata la mappa genetica
dello York Chocolate per tutte le
varianti previste dallo standard, sia per gli alleli espressione dei fenotipi
che per quelli recessivi. Questo
meticoloso lavoro di classificazione del patrimonio genetico dello
York Chocolate, oltre a caratterizzare scientificamente la razza, ha
permesso un completo controllo da parte degli allevatori delle espressioni
fisiche che verranno trasmesse ai vari discendenti. Classificazione dei geni e alleli correlati all’espressione fisica
dello York Chocolate Legenda: Geni
associati alla conformazione del corpo
Geni
associati alla conformazione del mantello The Sphinx gene. *The Longhaired gene à 1)“L” di tipo dominante e selvatico e` espressione di un mantello normale a pelo corto 2) “l” di tipo recessivo e mutante e` espressione di un mantello a pelo lungo o semi-lungo (Colonna 1 nelle tabelle seguenti). The Cornish Rex gene. The Devon Rex gene. The Oregon Rex gene. *The York under coat gene à 1) “Yuc” di tipo dominante e mutante e` espressione di un mantello senza sottopelo. 2)
“yuc”
di tipo recessivo e selvatico e` espressione di un mantello con sottopelo
piu` o meno fitto (Colonna 2 nelle tabelle seguenti). Geni
associati al colore del mantello *The Color Gene à 1) “B” Dominante espressione del colore nero 2)”b” recessivo rispetto a “B” espressione del colore marrone 3) “bl” recessivo rispetto a “B” e a “b” espressione del colore cannella (Colonna 3 nelle tabelle seguenti). The Orange-Making Gene. *The Color-Density Gene: 1)”D” Dominante espressione della non diluizione del colore 2) “d” recessivo espressione della diluizione del colore (Colonna 4 nelle tabelle seguenti). *The Albinism Gene à: 1)”C” Dominante espressione di un colore pieno ed uniforme sul mantello. 2)”cb” (Burmese) recessivo rispetto a “C”, produce un delicato albinismo riducendo il colore nero ad un marrone molto scuro. 3)”cs” (siamese) ) recessivo rispetto a “C” e codominante con “cb”, produce un albinismo intermedio riducendo il colore nero ad un beige chiaro con zone nere come il classico mantello siamese. 4)”ca” (albinismo occhi blu) recessivo rispetto a “C”, “cb” e “cs” produce un albinismo quasi completo che e` espressione di un mantello bianco traslucido associato ad occhi azzurri molto chiari. 5)”c” (albinismo completo) recessivo rispetto a “C”, “cb” ,“cs” e “ca” produce un albinismo completo che e` espressione di un mantello bianco traslucido associato ad occhi rosa (Colonna 5 nelle tabelle seguenti). *The Agouti Gene à: 1)”A” Dominante espressione di un mantello non uniforme. 2) “a” Recessivo espressione di un mantello uniforme (Colonna 6 nelle tabelle seguenti). The Tabby Genes. *The Color-Inhibitor Gene à: 1) “I” dominante e` espressione di una scolorazione della parte inferiore del pelo del mantello. 2) “i” recessivo e` espressione di una distribuzione uniforme del colore lungo tutto il pelo del mantello (Colonna 7 nelle tabelle seguenti). *The Spotting Gene à: 1) ”S” dominante produce delle macchie bianche completamente coprenti sul mantello di dimensione, forma e disposizione casuale. 2) ”Sp” e` una variazione di “S” produce una colorazione bianca coprente che, partendo da naso come V rovesciata corre lungo tutto il mento, il collo, il petto, la pancia ed anche parzialmente le zampe. Un’ulteriore variazione di questo allele, si presenta come un’unica macchia bianca di dimensione variabile sul petto. 3)”Sb” e` una variazione di “S” produce una colorazione bianca coprente confinata esclusivamente alle zampe. 4) “s” recessivo rispetto ad “S” e alle sue possibili variazioni “Sp” ed “Sb”, e` espressione della totale mancanza di macchie bianche sul mantello (Colonna 8 nelle tabelle seguenti). *The Dominant-White Gene à: 1) ”W” Dominante e` espressione di un mantello completamente bianco traslucido associato ad un colore degli occhi blu profondo o arancione. 2) ”Wv” (Van) Variazione di “W”, e` espressione di un mantello bianco traslucido con una leggera colorazione sulla calotta della testa e sulla punta delle orecchie. 3)”w” recessivo rispetto a “W” e alla sua possibile variazione “Wv” permette la piena espressione del colore (Colonna 9 nelle tabelle seguenti): Tabelle
di tutte le combinazioni di alleli dei geni espressione di tutte le sue
possibili varianti dello York Chocolate Le
colonne, formate di due celle ognuna, classificano ogni gene espressione fisica
dello York Chocolate. Nella prima riga sono indicati tutti gli alleli che
controllano il fenotipo, nella seconda riga tutti gli alleli associati
recessivi. Nelle tabelle, gli alleli in grassetto rilevano gli alleli che
possono diversificarsi all’interno della stessa variante di York
Chocolate. York
Chocolate Lavanda Solido:
York Chocolate Cioccolato Solido: PHENOTYPE l Yuc b D C a i s w RECESSIVE l Yuc b d C a i s w .
PHENOTYPE l Yuc b D C a i s w RECESSIVE l Yuc b D C a i s w
York Chocolate Bicolore Lavanda Particolor: PHENOTYPE l Yuc b d C a i Sp w RECESSIVE l Yuc b d C a i s w
.
PHENOTYPE l Yuc b d C a i Sp w RECESSIVE l Yuc b d C a i Sp w York Chocolate Bicolore Cioccolato Particolor:
.
.
PHENOTYPE l Yuc b D C a i Sp w RECESSIVE l Yuc b d C a i Sp w .
PHENOTYPE l Yuc b D C a i Sp w RECESSIVE l Yuc b D C a i Sp w . .
. Referenze Fulvio
Bresciani Nato a Saluzzo il 12/12/1960, si
e` laureato al Politecnico di Torino in Ingegneria Nucleare nel Febbraio
del 1985. Da oltre 15 anni lavora nel campo della ricerca scientifica in
particolare sull’ottica coerente e sulla teoria dei segnali applicata al
settore aerospaziale. E` autore di una serie d’articoli scientifici pubblicati
in campo nazionale ed internazionale. Da qualche anno studia modelli matematici
di genetica quantitativa per applicazione nell’ottimizzazione di funzioni a
multivariabile in campo non lineare. Di recente, in Collaborazione con
l’Università` di Pisa, sta sviluppando modelli matematici di genetica
quantitativa per la predizione di fattori ereditari nel campo della selezione
felina. In parallelo a quest’attività` sta lavorando sull’individuazione
del gene che regola nei gatti l’espressione del sottopelo. Bibliografia [1]
Feline Genetics Autori: R. Roger Breton, Nancy J Creek – Edito
(netpets 199 9) .
Negli anni passati la razza, a
causa di una certa superficialità da parte di alcuni allevatori americani, ha
rischiato di perdere alcune delle peculiarità fenotipiche che fin dalle origini
l’avevano caratterizzata per bellezza ed originalità. La
conseguente perdita di consensi da parte del pubblico, che sempre di più
vedevano nello York un gatto di casa e non quello stupendo felino la cui
bellezza spiccava nelle mostre fra tutti, ha rischiato di far definitivamente
estinguere la razza. In
particolare, uno dei danni a cui ancora oggi si sta cercando attraverso un
ferreo programma di selezione di porre rimedio, è stato l’introduzione nel
patrimonio genetico dello York del bicolore cosiddetto Spotting (presenza di
bianco sul mantello senza una delineata conformità). Questo è stato da
imputare a programmi di “out-crossing”
(incroci con gatti di casa senza pedigree) con soggetti non accuratamente
selezionati. Janet Chiefari, la prima selezionatrice di questa razza, ha avuto
tra gli altri, il grandissimo merito di stabilizzare nei soggetti bicolore la
conformazione del bianco secondo una geometria chiamata dai genetisti
“Particolor”. Lo
Spotting, come ben gli allevatori sanno, è una brutta bestia da gestire,
infatti, da padre a figlio non si sa mai come si tramandi e con quale
conformazione ed in quale quantità si possa presentare sul mantello. Questo
problema nello York, grazie a Janet, era stato completamente superato, una dura
selezione programmata le aveva permesso di ottenere soggetti bicolore sempre
uguali la cui splendida conformazione del bianco si trasmetteva nei discendenti
nella medesima quantità e disposizione. L'
"OUTCROSSING", come si sa, assolutamente necessario nelle
razze recenti a garantire un patrimonio genetico il più eterozigote possibile,
ha avuto da parte di questi allevatori un ulteriore scopo, quello di introdurre
una nuova conformazione nel mantello dello York, il cosiddetto “Van”. Il
Van, è una particolare espressione del mantello che si manifesta attraverso una
colorazione completamente bianca esclusa alcune piccole zone della nuca e della
punta delle orecchie che possono assumere colori diversi. Anche il Van, come il
Particolor, è un’espressione del mantello le cui leggi di ereditabilità sono
oggetto di discussioni tra i genetisti. Alcuni ritengono, questa, una variazione
dell’allele del gene “White”, altri invece una delle infinite
conformazioni attraverso il quale si può presentare l’allele spotting del
gene White-Spotting. Il tentativo di introdurre nel patrimonio genetico dello
York il “Van”, si e` tradotto in un vero disastro, ovvero, nei discendenti
bicolore di questi nuovi arrivi, il bianco ha cominciato a presentarsi nelle più
svariate conformazioni e quantità rendendo
impossibile ogni tentativo di controllo. Attraverso Anna Baldi, unica
selezionatrice Europea di York, si è riusciti in un passato recente, ad
importare dagli Stati Uniti, tra gli altri, uno di questi ormai rarissimi
soggetti bicolore aventi la classica conformazione Particolor. Accoppiandolo
successivamente con individui dalla colorazione solida, e per la gioia di noi
tutti amanti dello York, questo splendido esemplare ha dato vita a nuovi
soggetti bicolore tutti rigorosamente Particolor, dimostrando così di aver
ereditato totalmente il patrimonio genetico selezionato nei gatti di Janet
Chiefari. Ora,
è nostra ferrea intenzione, cercare di porre rimedio, almeno qui in Europa, ai
misfatti d’oltre Oceano, riportando lo York al suo antico splendore operando
in modo sistematico e sidergico. A
partire dagli allevatori, che in ogni caso dovranno cercare nel tempo di
estinguere la linea del bicolore rappresentato dal bianco disposto
“randomicamente” sul mantello, attraverso la definizione di uno nuovo
standard europeo molto selettivo, che bandisca ogni tipo di bicolore che si
allontani anche solo minimamente dalle caratteristiche del particolor
e che del resto non dovrà far altro che ricalcare ciò che è già
descritto negli standard CFF e CCA e per finire con l’aiuto dei giudici che,
nelle varie mostre ed esibizioni, dovranno essere ferrei nell’emettere giudizi
assolutamente in linea con lo standard senza eccezione alcuna.
IL VAN:
Dall'incrocio
di bicolori normali si sono presentati gattini con la caratteristica
comune di essere prevalentemente bianchi, con alcune macchie di colore
esclusivamente sul muso e sulla coda. Gli allevatori d'oltremare hanno stilato
uno standard provvisorio, in attesa che questi nuovi colori vengano immessi
nello standard originario. Attualmente negli Stati Uniti, i gatti
rispondenti alle caratteristiche sottoelencate vengono giudicati nella categoria
AOV (Colori non Riconosciuti). VAN
CIOCCOLATO E' un gatto prevalentemente
bianco, con il color cioccolato confinato sulle estremità: testa, coda e
qualche macchia ammessa sulle zampe. Uno o due spots (ciascuno non più largo di
7 cm nella sua parte più larga) sono permessi sul corpo. I gatti che mostrano
più di due macchie dovranno essere giudicati nella categoria dei bicolori. VAN
LAVANDA E' un gatto prevalentemente
bianco, con il color lavanda confinato sulle estremità: testa, coda e qualche
macchia ammessa sulle zampe. Uno o due spots (ciascuno nn più largo di 7 cm
nella sua parte più larga) sono permessi sul corpo. I gatti che mostrano più
di due macchie dovranno essere giudicati nella categoria dei bicolori.
Descripción general: Se trata de una nueva raza originaria de Estados Unidos, de donde toma el nombre: "York" por New York, y "Chocolate" por el color de su pelo. Se admite también su dilución, lilac, y las variantes bicolores de ambos colores. Es un gato exclusivamente de pelo semilargo. Esta raza aparece en los años 80. Sus origenes no están muy claros, pero parece que se trata de una raza procedente de gatos de pelo largo con antecedentes siameses Thai. La cabeza es casi redonda, frente ancha, cráneo redondeado y perfil ligeramente incurvado. Las orejas son de base ancha, bien separadas y bastante grandes. El mentón alineado con la nariz. El cuerpo es de tipo medio, entre siamés y extranjero (foreing), y no tiene que ser pesado. Bien musculado, las patas son bastante largas y musculadas. Los pies son redondos y pequeños. La cola tiene la base ancha, terminada en punta. El pelo es largo sobre los flancos, la espalda y la cola. Puede tener collar, en función de la época del año. El color del pelo es puro, de tonos marrones (Chocolate) y gris ligeramente rosado (Lilac). Estos mismos colores pueden combinarse con blanco, en una proporción de entre 1/3 y 1/2 de blanco para los bicolores. Los cachorros y jóvenes pueden tener marcas tabby (ghostmarking) hasta el año o año y medio, o una ligera coloración del pelo parecida al tipping. La textura es suave y sedosa.
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-amino
acid, is present in the reproductive tract of several mammals and recently
high concentrations have been found in spermatozoa, seminal plasma and
epididymal fluid of domestic cat (Buff et al 2001). The results indicated
that after thawing, sperm motility was better preserved in the presence of
taurine (25 mM: 52.2±5.9%; 50 mM: 53±3.7%) compared to in its
absence (23.8±4.6%), with no differences between the two concentrations
of taurine added.
