Elsevier

Small Ruminant Research

Volume 149, April 2017, Pages 91-98
Small Ruminant Research

Effect of the type of egg yolk, removal of seminal plasma and donor age on buck sperm cryopreservation

https://doi.org/10.1016/j.smallrumres.2017.01.007Get rights and content

Highlights

  • Egg yolk type, seminal plasma and donor age on buck sperm cryopreservation.

  • Buck spermatozoa washing is needed for cryopreservation.

  • Powdered egg yolk is recommended to reduce risk of microbiological contamination.

  • Clarified fresh egg yolk don’t show advantages and complicate extender preparation.

  • Animals of over two years old are recommended in creating a sperm bank.

Abstract

This study was designed to assess the effect of different types of egg yolk used in buck sperm cryopreservation and its concentration depending on the presence or absence of seminal plasma on sperm freezability from males of one and two years old. The aim was to reduce the potential risk of microbiological contamination of the extenders by replacement of fresh by pasteurized powdered egg yolk, and also to study the effect of the clarified fresh egg yolk, obtained by centrifugation of fresh egg yolk twice at 10.000 × g for 45 min at 4 °C. Briefly, fresh ejaculates (n = 6) from six to eight bucks were collected by artificial vagina in two consecutive autumns. After collection, ejaculates were mixed and split into two samples. One sample was washed twice by centrifugation at 600 × g for 10 min. Then, the pellet was split into three equal aliquots and re-suspended in an extender containing 15% (v/v) of different type of egg yolk (powdered, fresh or fresh clarified) supplemented with 5% glycerol (v/v) in Tris-based medium. The other semen sample was directly split into three equal aliquots and re-suspended in the same extenders, but containing only 2% (v/v) of different type of egg yolk (powdered, fresh or clarified). All samples were chilled for 4 h at 5 °C and then frozen to −196 °C. The results indicated that there was not superiority on the protective capacity of the different egg yolk types, either at 15 or 2% concentration on respective samples in post-thaw sperm analysis, regardless of donor age. Analyzed sperm parameters also showed better results in the washed samples than in the non-washed samples irrespective of the type of extender used and donor age. The use of clarified egg yolk did not offer any benefit to the fresh egg yolk, thus complicating the preparation process of such preservation media. Therefore, washing procedure of buck spermatozoa and the use of powdered egg yolk as a replacement for fresh egg yolk may be recommended for cryopreserving buck spermatozoa due to its efficiency and safety measures.

Introduction

The fresh egg yolk is a common component in sperm cryopreservation media and ever since it was used by Phillips Lardy in 1940, it has been considered a good non-penetrating cryoprotectant (reviewed by Marco-Jiménez et al., 2004). It has also been reported to protect the sperm cells from cold shock, by maintaining its sperm motility, acrosome and mitochondrial membrane integrity (Salamon and Maxwell, 2000). Such protection is basically determined by the low density lipoprotein (LDL) (Watson and Martin, 1975, Moussa et al., 2002), although the precise mechanism by which LDL protect sperm during freezing and thawing has not been clearly established. The addition of the LDL rich fraction obtained from fresh egg yolk by dialysis process (Moussa et al., 2002) or ultrafast centrifugation (Tonieto et al., 2010) may benefit post-thaw viability of the sperm.

However, fresh egg yolk being an animal product presents a biohazard potential risk because it may contain microbiological and another kind of contaminant that may compromise sperm quality. Therefore, the possibility of replacing the fresh egg yolk by a more in-noxious component with similar and even better sperm protection capacity from damage as a result of freezing is highly desirable. One such alternative could be the powdered egg yolk due to its pasteurization processes during production which eliminates possible bacterial effects (Marco-Jiménez et al., 2004). Despite this alternative, the high temperatures required during pasteurization can alter the composition of the egg yolk and as a result, reduce its protective capacity or even affect the quality of the sperm during preservation.

Furthermore, the sperm cryopreservation in bucks still remains a challenge because of the peculiarity observed in this species − its semen contains lipases from the secretions of the bulbourethral glands that interact with the egg yolk thus producing toxic substances that affect sperm cells (Iritani et al., 1964), and reducing the freezing ability (Leboeuf et al., 2000). To minimize the deleterious effects, sperm washing by centrifugation is usually done in order to remove the seminal plasma. Nevertheless, it has been shown that this practice may have a negative effect on sperm quality (Azeredo et al., 2001). Since the presence of some seminal plasma proteins in ram has been also reported to protect spermatozoa from damage caused by thermal shock (Colás et al., 2009) and important for proper functionality of sperm cells (Cardozo et al., 2006), the removal of seminal plasma may have adverse effect on such sperm cells.

Literature has also shown that semen quality varies depending on factors such as age, geographical location, the season and breed (Bongso et al., 1982, Chemineau, 1986). An example of age factor amongst breed of goats is observed in the Alpina goat producing good semen quality at six months of age while the Damascus (Al-Ghalban et al., 2004) or Murciano-Granadina (Roca et al., 1991) breeds produces such at a much older age.

Therefore, this study was designed to assess the effect of different types of egg yolk used in buck sperm cryopreservation and its concentration depending on the presence or absence of seminal plasma on sperm freezability from males of one and two years old, in order to reduce the potential risk of microbiological contamination of the extenders by replacement of fresh by pasteurized powdered egg yolk, and also to study the effect of the clarified fresh egg yolk.

Section snippets

Materials and methods

All chemicals and reagents were purchased from Sigma Chemical Co. (St. Louis, Mo, USA) unless otherwise stated. The powdered egg yolk was obtained from NIVE (Nunspeet Holland Eiproducten).

Results

Sperm viability estimated by E/N of the mixture of fresh goat ejaculates was similar (P > 0.05) among semen donors of one (76.8 ± 4.3) and two years old (81.1 ± 4.4), as well as the percentage of abnormal forms (1.4 ± 0.45; 2.4 ± 0.8) in semen from animals of one and two years old, respectively. In contrast, the volume (1.8 ± 0.1; 1.0 ± 0.1) and mass motility (3.8 ± 0.2; 2.5 ± 0.2) were significantly higher (P < 0.0001) in the older animals of two years compared to one years old, respectively. Similarly, after the

Discussion

The analysis of fresh semen quality of Blanca de Rasquera bucks showed that the age of the male had an effect on the volume and mass motility of the ejaculates and on the functional integrity of the sperm membrane with higher values seen at two years old males. This is in agreement with studies reported on Murciano-Granadina (Roca et al., 1991) and Damascus bucks (Al-Ghalban et al., 2004). Other reports on different species such as bulls (Bhakat et al., 2011, Brito et al., 2012) and rams (

Conflicts of interest

None.

Acknowledgements

The authors thank to the IRTA (Institut de Recerca i Tecnologia Agroalimnetàries, Spain) in Caldes de Montbui for their help with taking care of the semen donors. This work was supported by INIA (Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Spain) (RZ2009-00008-00-00), Generalitat de Catalunya (Catalonian Government, Spain) (2009SGR0621) and Fundación Carolina (Spanish Government, Spain).

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