Factors affecting motion characteristics of frozen‐thawed stallion spermatozoa

Abstract

Summary Five experiments were conducted to evaluate damage incurred in each processing step for cryopreservation of stallion spermatozoa. In Experiment 1, semen was centrifuged for 9 centrifugation times and the percentage of spermatozoa recovered after each treatment was calculated and spermatozoal motion characteristics analysed. Recovery of spermatozoa was ≥80% when spermatozoa were centrifuged for ≥10 min. Experiment 2 evaluated spermatozoa cryopreserved at 5 different concentrations in each of 2 extenders (skim milk‐egg yolk‐glycerol, SM‐EYG; and lactose‐EDTA, LAC). In SM‐EYG, TMOT and PMOT were higher at spermatozoal concentrations of 20, 200 and 400 times 106/ml (51%/41%, 52%/44%, 50%/43%, respectively) than for samples frozen at ≥800 times 106 spermatozoa/ml (41%/35%, 32%/27%; P<0.05). Spermatozoa frozen in LAC at a concentration of 20 times 106/ml resulted in the highest TMOT and PMOT (43% and 30%, respectively, P<0.05). The effect of freezing rate on motion characteristics of spermatozoa was evaluated in Experiment 3. The VCL of spermatozoa frozen in SM‐EYG was the only parameter affected by freezing rate (P<0.05). Experiment 4 evaluated motion characteristics after cryopreservation of spermatozoa in different sized straws (0.5 or 2.5 ml) in each of 2 extenders (SM‐EYG and LAC). In SM‐EYG, PMOT (38%) and VCL (109 μm/s) were highest when spermatozoa were frozen in 0.5 ml straws (P<0.05). In Experiment 5, spermatozoa thawed immediately after cryopreservation or thawed after storage in liquid nitrogen for 24–48 h were evaluated. There was no effect of length of storage in liquid nitrogen on spermatozoal motion characteristics (P<0.05). Experiment 6 evaluated the effects of cooling time to 5°C (0, 2.5 and 5 h) on motion characteristics of spermatozoa cryopreserved in 2 extenders (SM‐EYG and LAC). TMOT and PMOT were effected by cooling time, and there was a cooling‐time‐by‐extender interaction (P<0.05). In SM‐EYG, TMOT and PMOT were higher if spermatozoa were cooled to 5°C prior to initiation of freezing than if freezing was initiated at 20°C (P<0.05). A suggested protocol for cryopreservation of stallion spermatozoa would include: 1) centrifugation at 400 g for 14 to 16 min; 2) extension at 23°C with SM‐EYG to 400 times 106 spermatozoa/ml; 3) cool to 5°C for 2.5 h; 4) package in 0.5 ml straws at 5°C; 5) freeze in liquid nitrogen vapour at −160°C; and 6) thaw for 30 s in 37°C water.

Characterisation of movement pattern and velocities of stallion spermatozoa depending on donor, season and cryopreservation

“…In consideration of all examined parameters, the comparison of both media suggest that the composition of the extender has only little effect on the motility of stallion spermatozoa. In studies conducted by Heitland et al (1996) VCL of stallion spermatozoa was highest when spermatozoa were frozen with skim milk egg yolk extender. This extender largely conforms to cryoextender G used in this study.…”

Viabilidad in vitro e in vivo de los espermatozoides congelados/descongelados del conducto deferente de Alpacas (vicugna pacos)

“…Además muchos factores como de los crioprotectores afectan la sobrevivencia de los espermatozoides a la descongelación, entre estos se tiene las tasas de enfriamiento y calentamiento, diferentes técnicas de empacamiento, concentración de espermatozoides en las dosis congeladas, donde los parámetros de motilidad individual y motilidad progresiva decrecen a la descongelación (Heitland et al 1996). La integridad de la membrana del espermatozoide puede ser evaluado por diferentes pruebas, tal como tinciones supravitales o marcadores fluorescentes, pero la prueba hipo-osmótica es una prueba simple y de fácil ejecución (Watson 1995;Neild et al 1999).…”

Efeito da concentração espermática e da metodologia de inseminação artificial com sêmen equino congelado

“…Heitland et al (1996) Concomitantemente à aceitação do uso de sêmen congelado por várias associações de raça, houve uma renovação e estímulo nas pesquisas sobre protocolos para congelação de sêmen de garanhões (ALVARENGA et al, 2005).…”

“…Alguns trabalhos analisando variáveis de cinética espermática e integridade dos compartimentos celulares apontaram que a criopreservação em concentrações reduzidas pode ser um procedimento benéfico para os espermatozóides de eqüinos (HEITLAND et al, 1996;LOOMIS & CLARK, 1998;CLULOW et al, 2008;NASCIMENTO et al, 2008).…”

“…De acordo com Melo (2005) (PAPA et al, 1989;ANGOLA et al, 1992;ARRUDA et al, 1993;HEITLAND et al, 1996;LEIPOLD et al, 1998;LOOMIS & CLARK, 1998;BLANES et al, 2005;CLULOW et al, 2008;NASCIMENTO et al, 2008 …”