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Caspases are crucial mediators of programmed cell death (apoptosis). Apoptosis can occur in spermatozoa during spermatogenesis or epididymal transit, as well as in ejaculated spermatozoa. A high proportion of apoptotic sperm would be a poor indicator of the freezability of a raw seminal sample. Alpaca spermatozoa are notoriously difficult to freeze successfully. Therefore, the objectives of this study were to study caspase activation during incubation (37°C) of fresh alpaca spermatozoa, as well as before and after cryopreservation, to gain some insight into the mechanisms behind the vulnerability of alpaca spermatozoa. Eleven sperm samples were incubated for 4 h at 37°C (Study 1), and 23 samples were frozen using an automated system (Study 2). Caspase-3/7 activation was assessed at 0,1,2,3, and 4 h in samples incubated at 37°C (Study 1); and before/after cryopreservation (Study 2) using CellEvent™ Caspase 3/7 Green Detection Reagent and flow cytometry. The proportions of alpaca spermatozoa with caspase-3/7 activated increased (p < 0.05) after 3-4 h of incubation at 37°C; however, caspase activation was similar before and after cryopreservation (36.2 ± 11.2% vs. 36.6 ± 33.7%, p > 0.05). The high standard deviation found after freezing could be explained by the existence of two subpopulations: one subpopulation where caspase-3/7 activation decreased during cryopreservation (from 36.6 ± 9.1% to 1.5 ± 2.2%), and the other subpopulation where caspase-3/7 activation increased after cryopreservation (from 37.7 ± 13.0% to 64.3 ± 16.7%). In conclusion, after 3-4 h of incubation, caspase-3/7 activation increased in fresh alpaca sperm, whereas cryopreservation affects alpaca sperm samples in different ways.

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Eighty-five sperm samples were cryopreserved and SYBR14/PI, MitoTracker Deep Red FM, FITC-PSA/PI and chlortetracycline were used for imaging flow cytometry evaluation of sperm viability, mitochondrial membrane potential (MMP), acrosome integrity and sperm capacitation, respectively. Sperm motility was also registered. Sperm motility (46.1 ± 7.7 vs. 24.1% ± 6.5%), sperm viability (49.8 ± 11.5 vs. 32.3% ± 9.6%) and high MMP (49.8% ± 12.4% vs. 34.9% ± 9.9%) decreased significantly (p < .05) during cryopreservation process, in contrast to acrosome-reacted in viable spermatozoa (1.0% ± 1.6% vs. 1.0% ± 1.0%) and sperm capacitation (10.0 ± 9.8 vs. 8.2% ± 12.4%) that were similar (p > .05) before and after cryopreservation. Positive correlations were found between sperm motility versus high MMP (r = .63), sperm motility versus sperm viability (r = .67) and sperm viability versus high MMP (r = .88). In conclusion, cryopreservation of alpaca spermatozoa is related to a decrease in sperm motility, sperm viability and high MMP, meanwhile acrosome integrity and sperm capacitation are not affected.

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Epididymal alpaca sperm represent an alternative model for the study of alpaca semen. The objective of this study was to characterize the normal values of some functional variables in epididymal alpaca sperm using imaging flow cytometry. Alpaca testicles (n=150) were processed and sperm were recovered from the cauda epididymides. Only 76 samples with acceptable motility and sperm count were considered for assessment by imaging flow cytometry. Acrosome integrity and integrity/viability were assessed by FITC-PSA/PI and FITC-PNA/PI. Mitochondrial membrane potential (MMP) was assessed by MitoTracker CMXRos and MitoTracker Deep Red FM. Lipid peroxidation was evaluated using BODIPY 581/591 C11. Results show that the mean values for acrosome-intact sperm were 95.03±6.39% and 93.34±7.96%, using FITC-PSA and FITC-PNA, respectively. The mean values for acrosome-intact viable sperm were 60.58±12.12% with FITC-PSA/PI and 58.81±12.94% with FITC-PNA/PI. Greater MMP was detected in 65.03±15.92% and 59.52±19.19%, using MitoTracker CMXRos and MitoTracker Deep Red FM, respectively. Lipid peroxidation was 0.84±0.95%. Evaluation of acrosome-intact and acrosome-intact viable sperm with FITC-PSA/PI compared with. FITC-PNA/PI or MMP with MitoTracker CMXRos compared with MitoTracker Deep Red FM were correlated (P<0.05). The MMP using MitoTracker CMXRos was the only variable correlated (P<0.05) with sperm motility (r=0.3979). This report provides a basis for future research related to alpaca semen using the epididymal sperm model.

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El estudio tuvo por objetivo evaluar el efecto de tres dilutores (Tris, Tes y leche descremada) en la criopreservación de espermatozoides obtenidos del epidídimo de alpaca. Previamente se determinó el efecto del tiempo entre el beneficio/castración y la recuperación de los espermatozoides del epidídimo. Se utilizaron 24 testículos de alpacas para la obtención de los espermatozoides directamente del epidídimo en una solución fisiológica buferada (PBS). La recuperación de espermatozoides se realizó a las 0, 35, 48 y 72 horas (6 testículos por grupo) y se evaluó la motilidad, concentración espermática e integridad funcional de membrana. Los espermatozoides recuperados a partir de 35 horas post beneficio/castración no fueron aptos para ser congelados. Las muestras recuperadas a las 0 horas fueron diluidas con Tris, Tes y leche descremada, enfriadas desde 35 a 5°C en 90 minutos, envasadas en pajillas de 0.25 ml y congeladas en nitrógeno líquido. Se evaluó la motilidad, integridad funcional de membrana y vitalidad/integridad acrosomal en las muestras descongeladas. La motilidad fue de 14.0, 8.6 y 17.0% en los grupos Tris, Tes y leche descremada, respectivamente, donde el grupo leche descremada fue significativamente mejor que el grupo Tes (p<0.05). Los porcentajes de integridad funcional de membrana y vitalidad/integridad acrosomal fueron similares entre los tres grupos. Se concluye que los tres dilutores brindan efectos similares para la criopreservación de espermatozoides epididimarios de alpaca.

The objective of this study was to evaluate the effect of three semen extenders (skim milk, Tris, and Tes) on cryopreservation of epididymal alpaca sperm. Previously, the effect of timespan since slaughtering or castration to the recovery of epididymal sperm was evaluated. Twenty-four alpaca testicles were used to obtain sperm from the epididymis in a buffered saline solution (PBS). The recovery of spermatozoa was performed at 0, 35, 48, and 72 hours (6 testes per group) after slaughtering or castration. Sperm motility, concentration, and sperm membrane functional integrity were analyzed. Sperm recovered after 35 hours was not usable for cryopreservation. Sperm samples recovered at 0 hours were subjected to the process of freezing. Samples were diluted with skim milk, Tris, and Tes, cooled from 35 to 5 °C in 90 minutes, packed into 0.25 ml straws and frozen in liquid nitrogen. After thawing, straws were evaluated by motility, sperm membrane functional integrity and vitality/acrosome integrity. Motility was 17.0, 14.0, and 8.6% on skim milk, Tris and Tes groups respectively, where the skim milk group was significantly better than the Tes (p<0.05). Percentages of sperm membrane functional integrity and vitality/acrosomal integrity were similar among the three extenders. It was concluded that all three extenders provided similar effects for the cryopreservation of epididymal alpaca spermatozoa.

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Se evaluó el efecto del tratamiento superovulatorio en las dos fases del ciclo ovárico sobre la respuesta folicular y la calidad embrionaria en 45 llamas hembras adultas. Se incluyeron en el estudio aquellos animales que a la ecografía presentaron un folículo preovulatorio >7 mm. Los animales se distribuyeron en tres grupos: T0 (no estimulado), T1 (tratamiento superovulatorio en fase no luteal) y T2 (tratamiento superovulatorio en fase luteal). Los animales de T1 y T2 recibieron 1 ml de LH (día 0) para sincronizar la onda folicular y 1000 UI de eCG (día 3) como tratamiento superovulatorio. Se utilizaron esponjas vaginales impregnadas con progesterona entre el día 3 y 7 para simular la fase luteal en el T2. La inducción de la ovulación se hizo mediante monta natural y aplicación de 1 ml de GnRH (día 8). La colección y evaluación de embriones se realizó 7 días post cópula (día 15) en T1 y T2. En el grupo T0 se realizó monta natural y aplicación de GnRH y 7 días después se realizó la colección de embriones. El número de folículos preovulatorios fue mayor en T1 (11.07 ± 7.53) y T2 (6.13 ± 7.11) con respecto a T0 (1.07 ± 0.26) (p<0.05). El número de cuerpos lúteos fue mayor en T1 (9.27 ± 3.37) con respecto a T0 (1.07 ± 0.26) y T2 (6.47 ± 4.29) (p<0.05). Asimismo, el número de embriones recuperados fue mayor en T1 (3.47 ± 4.26) con respecto a T0 (0.33 ± 0.48) y T2 (1.33 ± 2.53). Los resultados permiten concluir que la aplicación del tratamiento superovulatorio durante una fase no luteal permiten obtener una mejor respuesta ovárica y embrionaria en comparación con tratamientos superovulatorios aplicados en fase luteal.

The effect of superovulatory treatment during the two phases of the ovarian cycle on follicular growth and embryo quality was evaluated in 45 sexually adult llamas. Animals bearing a >7 mm follicle, observed by ultrasonography, were selected and allocated into 3 groups: T0 (non-stimulated), T1 (superovulatory treatment during the non luteal phase), and T2 (superovulatory treatment during the luteal phase). Animals in groups T1 and T2 received 1 ml of LH (day 0) for synchronization of the follicular wave and 1000 IU of Ecg (day 3) as superovulatory treatment. Vaginal sponges impregnated with progesterone were used on days 3 to 7 in T2 to simulate the luteal phase. The induction of the ovulation (day 8) was done through natural mating and the application of GnRH (1 ml). Embryo recovery was done 7 days after natural mating (day 15) on T1 and T2. Similarly, embryo recovery was done 7 days after natural mating and application of GnRH in T0. The number of preovulatory follicles was larger in T1 (11.07 ± 7.53) and T2 (6.13 ± 7.11) than in T0 (1.07 ± 0.26) (p<0.05). The number of corpora lutea was larger in T1 (9.27 ± 3.37) than in T0 (1.07 ± 0.26) and T2 (6.47 ± 4.29) (p<0.05). The number of recovered embryos was larger in T1 (3.47 ± 4.26) than in T0 (0.33 ± 0.48) and T2 (1.33 ± 2.53). The results showed that superovulatory treatment during the non luteal phase had a better response than superovulatory treatment during the luteal phase.

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