Motility and recovery of alpaca (Vicugna pacos) spermatozoa after centrifugation in a density gradient solution

Document Type: Full paper (Original article)


1 Department of Animal Production, Universidad Nacional Agraria La Molina, Lima, Perú; Faculty of Veterinary Medicine and Zootechnics, Universidad Nacional Micaela Bastidas de Apurímac, Abancay, Perú

2 Department of Animal Production, Universidad Nacional Agraria La Molina, Lima, Perú

3 Faculty of Veterinary Sciences, Universidad de Buenos Aires, CONICET, Argentina

4 Fundo Mallkini de Michael & Cia S.A., Azángaro, Perú

5 Vivanco International SAC, Lima, Perú

6 Department of Animal Science, College of Agriculture and Life Science, Iowa State University, Ames, Iowa, 50011, USA


Background: One of the factors limiting successful processing of alpaca (Vicugna pacos) semen is the viscosity of seminal plasma. The viscous nature of the collected ejaculate has hindered sperm cryopreservation as well as artificial insemination (AI) under field conditions. Aims: The objective of this investigation was to evaluate recovery, motility, and plasma membrane integrity of alpaca spermatozoa after centrifugation in one of two different solutions at one of three different combinations of speed and time. Methods: A total of 24 ejaculates was recovered from seven reproductively sound Huacaya males using a modified artificial vagina (AV) after training the animals for semen collection. A 2 × 3 factorial treatment arrangement was utilized for this study. Ejaculates were divided into fractions for centrifugation in one of two solutions (Tris extender or PureSperm®80 density gradient solution) at one of three combinations of speed and time (492 × g for 15 min, 1968 × g for 10 min, or 4448 × g for 7 min). The experiment was replicated eight times. Results: Analysis revealed that centrifugation at 4448 × g for 7 min in PureSperm80 provided a high recovery rate of spermatozoa with the highest sperm motility and functional integrity of plasma membrane post-centrifugation. Conclusion: Results suggest that adoption of this procedure (centrifugation at 4448 × g for 7 min in PureSperm80) in the initial processing of alpaca ejaculates may enhance subsequent ability to use semen for AI and other assisted reproductive biotechnologies in this species.


Adams, GP; Ratto, MH; Collins, CW and Bergfelt, DR (2009). Artificial insemination in South American camelids and wild equids. Theriogenology. 71: 166-175.

Alarcon, V; Garcia, W and Bravo, W (2012). Artificial insemination of alpacas with semen collected by vaginal aspiration and by artificial vagina. Rev. Inv. Vet. Peru. 23: 58-64.

Barati, F; Khaksary Mahabady, M and Mohammadi, Gh (2009). Cryopreservation of in situ cool stored buffalo (Bubalus bubalis) epididymal sperm. Iran. J. Vet. Res., 10: 339-345.

Boomsma, CM; Heineman, MJ; Cohlen, BJ and Farquhar, C (2007). Semen preparation techniques for intrauterine insemination. Cochrane Database of Systematic Reviews. Issue 4. Art. No.: CD004507. doi: 10.1002/14651858. CD004507.pub3.

Bravo, PW; Callo, M and Garnica, J (2000b). The effect of enzymes on semen viscosity in llamas and alpacas. Small Rum. Res., 38: 91-95.

Bravo, PW; Flores, U; Garnica, J and Ordoñez, C (1997a). Collection of semen and artificial insemination of alpacas. Theriogenology. 47: 619-626.

Bravo, PW; Flores, U and Ordoñez, C (1997b). Effect of repeated collection on semen characteristics of alpacas. Biol. Reprod., 57: 520-524.

Bravo, PW; Skidmore, JA and Zhao, XX (2000a). Reproductive aspects and storage of semen in Camelidae. Anim. Reprod. Sci., 62: 173-193.

Campos, ACN; Nunes, JF; Monteiro, AWU; Figueirêdo, EL; Pinheiro, JHT; Ferreira, MAL and Araújo, AA (2004). Viability of washed and unwashed goat sperm diluted in coconut water, cooled and stored at 4°C. Rev. Bras. Ciênc. Vet., 11: 178-182 (in Portuguese).

Claassens, OE; Menkveld, R and Harrison, KL (1998). Evaluation of three substitutes for Percoll in sperm isolation by density gradient centrifugation. Hum. Reprod., 13: 3139-3143.

Dávalos, R and Olazábal, J (2002). Evaluación de dos formas de colección de semen en alpacas. Rev. Inv. Vet. Perú., 13: 98-99 (in Spanish).

Dorado, J; Alcaraz, L; Gálvez, MJ; Acha, D; Ortiz, I; Urbano, M and Hidalgo, M (2013). Single-layer centrifugation through PureSperm®80 selects improved quality spermatozoa from frozen-thawed dog semen. Anim. Reprod. Sci., 140: 232-240.

EL-Bahrawy, KA (2010). Effect of seminal plasma centrifugation for viscosity eliation on cryopreservation of dromedary camel semen. Nat. Sci., 8: 196-201.

Fernandez-Baca, S and Calderon, W (1965). Metodos de colección de semen de la alpaca. UNMSM J. Fac. Vet. Med., 18, 19, 20: 13-17 (in Spanish).

Fumuso, FG; Carretero, MI; Neild, D; Miragaya, M and Giuliano, SM (2015). Evaluación de la viabilidad y el estado acrosomal de espermatozoides de llama (Lama glama). Resultados preliminares. Primer Congreso de la Sociedad Latinoamericana de Reproducción Animal, 2015, Buenos Aires, Argentina. P: 1 (in Spanish).

Garcia, W; Alracon, V and Bravo, W (2017). Artificial insemination of alpacas with refrigerated semen and the inclusión of two types of egg yolk. Rev. Inv. Vet. Perú., 28: 337-344.

Garnica, J; Achata, R and Bravo, PW (1993). Physical and biochemical characteristics of alpaca semen. Anim. Reprod. Sci., 32: 85-90.

Giuliano, J; Carretero, M; Gambarotta, M; Neild, D; Trasorras, V; Pinto, M and Miragaya, M (2010). Improvement of llama (Lama glama) seminal characteristics using collagenase. Anim. Reprod. Sci., 118: 98-102.

Gomes-Alves, S; Alvarez, M; Nicolas, M; Martínez-Rodríguez, C; Borragán, S; Chamorro, CA; Anel, L and de Paz, P (2014). Salvaging urospermic ejaculates from brown bear (Ursus arctos). Anim. Reprod. Sci., 150: 148-157.

Gómez-Quispe, OE; Pérez, MG and Ojeda-Gutierrez, YG (2016). Survival to freezing of sperm collected by vas deferens from Vicugna pacos with Tempol antioxidant addition. Asian J. Anim. Vet. Adv., 11: 377-382.

Hernández-López, L; Umland, N; Mondragón-Ceballos, R and Nayudu, PL (2005). Comparison of the effects of Percoll and PureSperm® on the common marmoset (Callithrix jacchus) semen. J. Med. Primatol., 34: 86-90.

Hollinshead, FK; Evans, G; Evans, KM; Catt, SL; Maxwell, WMC and O’Brien, JK (2004). Birth of lambs of a pre-determined sex after in vitro production of embryos using frozen-thawed sex-sorted and re-frozen-thawed ram spermatozoa. Reproduction. 127: 557-568.

Jeyendran, R; Van Der Ven, H; Perez-Pelaes, M; Crabo, B and Zaneveld, L (1984). Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics. J. Reprod. Fertil., 70: 219-228.

Juyena, NS; Vencato, J; Pasini, G; Vazzana, I and Stelletta, C (2013). Alpaca semen quality in relation to different diets. Reprod. Fertil. Dev., 25: 683-690.

Kershaw-Young, CM and Maxwell, WM (2011). The effect of seminal plasma on alpaca sperm function. Theriogenology. 76: 1197-1206.

Kovács, A and Foote, RH (1992). Viability and acrosome staining of bull, boar and rabbit spermatozoa. Biotech. Histochem., 67: 119-124.

Lichtenwalnerl, AB; Woods, GL and Weber, JA (1996). Seminal collection, seminal characteristics and pattern of ejaculation in llamas. Theriogenology. 46: 293-305.

Lima, ICS; Andrade, IRA; Aguiar, GV; Silva, MM; Catunda, AGV; Martins, GA; Gadelha, CRF and Campos, ACN (2013). In vitro evaluation of goat cauda epididymal sperm, cooled in different extenders at 4°C. Arch. Zootec., 62: 429-437.

Malo, C; Crichton, EG; Morrell, JM; Pukazhenthi, BS; Johannisson, A; Splan, R and Skidmore, JA (2018). Colloid centrifugation of fresh semen improves post-thaw quality of cryopreserved dromedary camel spermatozoa. Anim. Reprod. Sci., 192: 28-34.

Malo, C; Crichton, EG and Skidmore, JA (2017). Optimization of the cryopreservation of dromedary camel semen: cryoprotectants and their concentration and equilibration times. Cryobiology. 74: 141-147.

Matás, C; Decuadro, G; Martínez-Miró, S and Gadea, J (2007). Evaluation of a cushioned method for centrifugation and processing for freezing boar semen. Theriogenology. 67: 1087-1091.

Maxwell, WMC; Parrilla, I; Caballero, I; Garcia, E; Martinez, EA; Vasquez, JM and Rath, D (2007). Retained functional integrity of bull spermatozoa after double freezing and thawing using PureSperm® density gradient centrifugation. Reprod. Dom. Anim., 42: 489-494.

Monteiro, GA; Guasti, PN; Hartwig, FP; Dellaqua, JAJÁ; Alvarenga, MA and Papa, FO (2013). Cooling of ejaculated and epididymal stallion sperm. Arq. Bras. Med. Vet. Zootec., 65: 681-686.

Morrell, JM; Dalin, AM and Rodriguez-Martinez, H (2009). Comparison of density gradient and single layer centrifugation of stallion spermatozoa: yield, motility and survival. Equine Vet. J., 41: 53-58.

Morton, K; Bathgate, R; Evans, G and Maxwell, WMC (2007). Cryopreservation of epididymal alpaca (Vicugna pacos) sperm: a comparison of citrate, - Tris- and lactose-based diluents and pellets and straws. Reprod. Fertil. Dev., 19: 792-796.

Morton, KM; Gibb, Z; Leahy, T and Maxwell, WMC (2012). Effect of enzyme treatment and mechanical removal of alpaca (Vicugna pacos) seminal plasma on sperm functional integrity. J. Camelid Sci., 5: 62-81.

Morton, KM; Vaughan, JL and Maxwell, WMCh (2008). The continued development of artificial insemination technology in alpacas. Rural Industries Research and Development Corporation. Publication No. 08/05, Project No. US-138A. Autralia. PP: 83-91.

Mosaferi, S; Niasari-Naslaji, A; Abarghani, A; Gharahdaghi, AA and Gerami, A (2005). Biophysical and biochemical characteristics of bactrian camel semen collected by artificial vagina. Theriogenology. 63: 92-101.

Niasari-Naslaji, A; Mosaferi, S; Bahmani, N; Gerami, A; Gharahdaghi, AA; Abarghani, A and Ghanbari, A (2007). Semen cryopreservation in Bactrian camel (Camelus bactrianus) using SHOTOR diluent: effects of cooling rates and glycerol concentrations. Theriogenology. 68: 618-625.

Niasari-Naslaji, A; Mosaferi, S; Bahmani, N; Gharahdaghi, AA; Abarghani, A; Ghanbari, A and Gerami, A (2006). Effectiveness of a tris-based extender (SHOTOR diluent) for the preservation of Bactrian camel (Camelus bactrianus) semen. Cryobiology. 53: 12-21.

Nicolas, M; Alvarez, M; Borragán, S; Martinez-Pastor, F; Chamorro, CA; Alvarez-Rodriguez, M; de Paz, P and Anel, L (2012). Evaluation of the qualitative and quantitative effectiveness of three media of centrifugation (Maxifreeze, Cushion Fluid Equine, and PureSperm 100) in preparation of fresh or frozen-thawed brown bear spermatozoa. Theriogenology. 77: 1119-1128.

Ortega-Ferrusola, C; González-Fernández, L; Muriel, A; Macías-García, B; Rodríguez-Martínez, H; Tapia, JA; Alonso, JM and Peña, FJ (2009). Does the microbial flora in the ejaculate affect the freezeability of stallion sperm? Reprod. Dom. Anim., 44: 518-522.

Pacheco, JI; Mamani, RH and Velez, VM (2017). Effect of
seminal plasma on hipoosmotic swelling test in fresh alpaca spermatozoa. MOJ Biol. Med., 2: 00059.

Pérez, MG; Quintano, J and Perez, UH (2014). Sperm survival in cooling a 5°C recovered the vas deferens of alpaca in three extenders with two membrane protector. Spermova. 4: 153-158.

Raymundo, F; Huanca, W; Huanca, T; Huerta, S and Cordero, A (2006). Efecto de tres dilutores en la conservación del semen de alpacas. Rev. Inv. Vet. Perú., 17: 125-130 (in Spanish).

Salvador, I; Yániz, J; Viudes-De-Castro, MP; Gómez, EA and Silvestre, MA (2006). Effect of solid storage on caprine semen conservation at 5°C. Theriogenology. 66: 974-981.

Santiani, A; Huanca, W; Sapana, R; Huanca, T; Sepulveda, N and Sanchez, R (2005). Effects on the quality of frozen-thawed alpaca (Lama pacos) semen using two different cryoprotectants and extenders. Asian J. Androl., 7: 303-309.

Scott, C; Torre, T; Souza, D; Mansano, M; dos Santos, C and Ferreira, F (2016). Viability of cryopreserved bull spermatozoa collected from the epididymis stored for 12 hours at 4°C. R. Bras. Ci. Vet., 23: 191-195 (in Portuguese).

Söderlund, B and Lundin, K (2000). The use of silane-coated silica particles for density gradient centrifugation in in-vitro fertilization. Hum. Reprod., 15: 857-860.

Soler, AJ; Perez-Guzmsoler, AJ; Perez-Guzman, MD and Garde, JJ (2003). Storage of red deer epididymis for four days at 5°C: effects on sperm motility, viability, and morphological integrity. J. Exp. Zoo., 295: 188-199.

Vásquez, J; Florentini, E and Valdivia, M (2012). Hypoosmotic swelling test in alpaca (Vicugna pacos) epididymal spermatozoa. Reprod. Dom. Anim., 47: 83-87.

Vivanco, HW; Ayala, JE; Huaman, E; Leon, S and Ponce, D (2010). Improvement of sperm evaluation through the innovation in the design of semen collection and the control of fixed and random effects in Huacaya alpacas of the Munay Paqocha Center. Technical report. Instituto Nacional de Innovación Agraria (INIA). Contract N°-228-2008-CONCYTEC-P. Lima, Perú. PP: 1-26.

Von Baer, A and Hellemann, C (1998). Semen variables in llama. Arch. Med. Vet., 30: 171-176.

Wani, NA; Billah, M and Skidmore, JA (2008). Studies on liquefaction and storage of ejaculated dromedary camel semen. Anim. Reprod. Sci., 109: 309-318.

WHO (2010). WHO Laboratory Manual for the Examination an Processing of Human Semen (5th Ed.), World Health Organization, Switzerland. ISBN-13:978-9241547789. P: 287.