Elsevier

Theriogenology

Volume 72, Issue 2, 15 July 2009, Pages 160-168
Theriogenology

Sperm characteristics and in vitro fertilization ability of thawed spermatozoa from Black Manchega ram: Electroejaculation and postmortem collection

https://doi.org/10.1016/j.theriogenology.2009.02.002Get rights and content

Abstract

The aim of this study was to assess two models of sperm collection on the quality and fertility of thawed spermatozoa from Black Manchega rams, a threatened breed. Sperm samples were collected by electroejaculation and postmortem from each male. Samples were diluted with Biladyl and frozen. Motility (subjective and objective by means of computer-assisted semen analysis), membrane integrity, and acrosomal status (microscopy) were assessed on fresh and thawed semen; plasmalemma integrity, mitochondrial membrane potential, DNA integrity, and acrosomal status were evaluated by flow cytometry on thawed semen. Thawed spermatozoa were used in a heterologous in vitro fertilization test. After thawing, the proportion of live spermatozoa with intact membrane (YO-PRO-1−/PI−) was higher for postmortem samples (P < 0.001), although the ratio of YO-PRO-1− spermatozoa within the PI− population was higher for ejaculated samples (P = 0.007). Likewise, the proportion of live spermatozoa having high mitochondrial membrane potential (MitoTracker+) and intact acrosomes (PNA−) was higher for postmortem samples (P < 0.001 and P < 0.001, respectively). Considering only live spermatozoa, the ratio of MitoTracker+/PNA− cells was higher for electroejaculated samples (P = 0.026 and P = 0.003). Both electroejaculated and postmortem samples fertilized oocytes. Nevertheless, electroejaculated samples yielded a higher percentage of hybrid embryos (P = 0.041). In conclusion, although postmortem spermatozoa had better sperm quality after thawing, electroejaculated spermatozoa showed higher ratios for sperm quality when only the live population was considered. Electroejaculated and postmortem samples might be used for germplasm banking of this threatened breed, but the fertility of postmortem spermatozoa might be lower.

Introduction

The Manchega sheep is an autochthonous dairy breed from Spain with a white and a black variety. Currently, there are few herds of Black Manchega sheep (around 2000 animals), and in Spain it is considered an endangered breed of special interest (RD 1682/1997). As recommended by the Food and Agriculture Organization (FAO) of the United Nations, endangered breeds must be preserved by ex situ and in situ means.

One of the ex situ procedures consists of the development of genetic resource banks, cryopreserving gametes and embryos, thus allowing to store genetic resources indefinitely [1]. Assisted reproductive technologies have been demonstrated to be successful tools to reproduce endangered species; sperm cryopreservation, in combination with artificial insemination, being the method that has been most extensively applied.

Semen can be collected from live animals by artificial vagina [2], but this technique requires a previous training period [3]. Electroejaculation is an alternative when males are not trained to artificial vagina. This method allowed the collection of high-quality semen in Guirra ram without noticeable differences from semen collected by artificial vagina [4]. In that study, sperm quality was higher for electroejaculated samples even after cryopreservation.

Postmortem recovery of spermatozoa and their use has received much less attention in sheep than in other domestic species [5], [6]. Collection and cryopreservation of epididymal spermatozoa is a useful method to rescue germplasm of dead animals that would be otherwise lost and use it to preserve endangered breeds. This kind of sample has been successfully cryopreserved and used for artificial insemination or in vitro fertilization in other ungulates, such as red deer [7], [8], [9], [10], [11], [12], [13].

Ejaculated spermatozoa have a lower cryosurvival and osmotic resistance than that of epididymal spermatozoa [14]. Nevertheless, it has been showed that adding seminal plasma to epididymal spermatozoa protects against the effects of cooling and freezing [15], [16]. However, seminal plasma can also have detrimental effects on cryosurvival [17].

In vitro fertility of fresh semen obtained by artificial vagina and postmortem has been studied in sheep [5]. However, to date no studies have been conducted in this species for evaluating the effect of collection method (electroejaculation or postmortem recovery) on postthawing sperm quality and fertility.

Considering this background, the objective of this study was to evaluate the postthawing sperm quality and in vitro fertility of sperm samples obtained both by electroejaculation and by postmortem collection from Black Manchega rams in order to assess its suitability for germplasm banking and the differences between both methodologies.

Section snippets

Animals and reagents

All animal procedures were performed in accordance with the Spanish Animal Protection Regulation RD 1201/2005, which conforms to European Union Regulation 2003/65. Six rams of the Black Manchega breed (age >3 yr) were used. Males came from a private farm, whose owner decided to sacrifice these animals within a genetic improvement program, a practice compatible with the protection plans for the breed. Rams were temporally maintained and managed at the Regional Center of Animal Selection and

Results

Sperm quality of fresh samples was not different between collection methods, except for sperm viability, which was higher in postmortem samples (P < 0.001) (Table 1).

After thawing, sperm motility and velocity were higher in postmortem samples (P = 0.043 and P = 0.001, respectively) (Table 1). There were significant differences between electroejaculated and postmortem samples for most of the parameters evaluated by flow cytometry, with lower values for electroejaculated spermatozoa (YO-PRO-1−/PI−, P < 

Discussion

In this study, we assessed the effect of collection method (electroejaculation and postmortem) on sperm quality for fresh, thawed, and incubated samples and on in vitro fertility from Black Manchega rams. Both collection methods provided good sperm quality. Electroejaculation is an excellent choice for animals that have not been trained to artificial vagina, whereas postmortem collection can be used as an emergency method when valuable males die suddenly.

Our results showed that sperm quality

Acknowledgments

This work was supported by the Education and Science Council (PBI-05-011), by the Spanish Ministry of Education and Science (RZ2006-00006-C3), and by the Agriculture Council (PREG-05-004) of Junta de Comunidades de Castilla-La Mancha (JCCM). The authors thank Frimancha Industrias Cárnicas S.A. for their collaboration in the collection of the ovaries used in this work. Olga García-Álvarez and Alejandro Maroto Morales were recipients of scholarships from INIA and JCCM, respectively. Felipe

References (37)

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