A comparison between in vitro conservation and in vivo fertilizer capacity of refrigerated rabbit semen with two diluents

1. Introduction

Rabbit farming in Mexico has had a limited development (Torres, 2012). In 2001, the Secretary of Agriculture, Livestock, Rural Development, Fisheries and Food (SAGARPA, for its acronym in Spanish) officially recognized rabbit breeding as a livestock activity and the National Information System initiated the statistical recording of it. Researches on rabbit breeding have been scarce in the country and have generated in a disjointed and dispersed manner, and more so, they have not taken advantage of the biological-economic benefits that the species offers, such as a high prolificacy, a short reproductive cycle and a large degree of genetic diversity (Lavara et al., 2003). Therefore, AI offers management advantages, since it allows to synchronize deliveries of a large number of animals, using a minimum number of stallions, reducing the risk of disease dissemination and permitting the use of refrigerated semen (Bilbao, 1996). One of the most important factors involved in AI is the medium in which the semen is diluted (Castellini, 1996), which will allow maintaining a higher proportion of live spermatozoa until the insemination moment (Vega et al., 2012). Unlike other species, rabbit spermatozoa have a low water permeability and a high energy activation coefficient; reasons why spermatic survival is inferior when using techniques such as cryopreservation. Therefore, the conservation of rabbit semen through refrigeration is a timely strategy to lengthen spermatic vitality by reducing the functionality alteration and the vitality loss when subjected to the freezing/unfreezing process. Hence, the objective of this work was to evaluate the effect of two diluents for seminal refrigeration (at 5°C) for 24 hours, on the rabbit spermatic quality, and the parameters of fertility and prolificacy of the females artificially inseminated with said refrigerated semen.

2. Material and methods

New Zealand white breed rabbits were used. To obtain the ejaculates, a French type artificial vagina (AV) was used at a 45°C temperature (Lavara et al., 2013). It was later transferred into a thermos with a 38°C temperature to the Laboratory of Biology of Reproduction at the Autonomous Metropolitan University-Iztapalapa.

2.1 Seminal evaluation

The samples were placed in a water bath (38°C). The mass motility was evaluated using a scale of 0 to 5, where 0 indicates absence of movement and 5, substantial rapid spermatic waves. For this, a sample of 5 μl was placed on a slide and evaluated under the optical microscope with a 10x objective. For progressive motility, a sample of 5 μl was placed on a slide and a coverslip was put, evaluating with a 20x objective, assessing the percentage of spermatozoa that exhibited progressive movement. To determine the percentage of vitality and morphology, eosin-nigrosin staining was used, in which live spermatozoa do not stain and the dead have rosacea. As for the morphology, they were categorized as normal or abnormal, when they presented head, middle piece and tail abnormalities. The spermatic concentration was determined using the Neubauer chamber; for this, a dilution of 995μl of distilled water and 5μl of semen was made, placing 15μl of it in each of the sections of the chamber and evaluating it under a microscope with a 40x objective. The result was expressed in millions of sperm per mL (X106/mL). The state of the acrosome was evaluated, setting 10 μl of the spermatic sample with Tris medium and 10 μl of chlortetracycline preparation (CTC), spreading it on a slide previously tempered at 37°C. The sample was observed with a 100X magnification, and a drop of immersion oil was placed to provide a sharper focus. Through the CTC-emitted fluorescence, the patterns and the acrosomal state were determined: pattern F = capacitated spermatozoa with an intact acrosome, pattern B = uncapacitated spermatozoa with an intact acrosome, and pattern RA = uncapacitated spermatozoa with acrosomal reaction.

2.2 Samples processing and evaluation

The volume of each ejaculate was divided into two aliquots. Each one was diluted in a 1:1 ratio (semen/Tris, v/v) in TEY or TSM diluents, using 1 mL Eppendorf tubes. Immediately afterwards they were refrigerated at 5°C for 24 hours. The cooling rate from room temperature to 5°C was 0.68°C/min. The determination of progressive motility, vitality and morphology was carried out at 2, 4, 6, 8, 12 and 24 hours after the refrigeration, and the determination of the acrosome state was carried out at 2, 8 and 24 hours after the refrigeration for both diluents used.

2.3 Artificial insemination

The female subjects were exposed to a sudden environmental change (they were moved to another cage) 8 hours before the artificial insemination, with the purpose of heat synchronization. Afterwards, the subject was placed inside a support and subjection device (known as "cannon"), exposing the hind limbs and the genital area to facilitate the insemination procedure (Alvariño, 2009). 0.5 ml was deposited per dose of the media used (TEY and TSM) at different post-refrigeration times (2, 8 and 24 hours, in addition to the control). Immediately after, 25 IU of a GnRH analogue were applied to induce ovulation (Fertagyl®). The number of inseminated females was 5 for each established medium and time (n=35).

2.4 Statistical analysis

The parameters of sperm quality (progressive motility, vitality, normal morphology and acrosomal integrity) were compared between treatments using ANOVA. Using the NCSS statistical package. A P value of less tan 0.05 (P <0.05) was considered significant.

3. Results

Out of the freshly collected ejaculates, a volume of 0.47 ± 0.21 ml was obtained, category 4 mass motility, progressive motility of 85 ± 4%, vitality of 88 ± 5%, normal morphology of 71 ± 7.13%, and a concentration of 283.81 ± 103.22 106/ml. Parameters of progressive motility, vitality and normal morphology around 24 hours after refrigeration with both diluents (Table 1). The integrity of the acrosome at 2, 8 and 24 hours after refrigeration is indicated in Table 2. In Table 3, the reproductive behavior of female rabbits inseminated with fresh semen and refrigerated semen around 24 hours, using both diluents, is shown.

Table 1. Progressive motility, vitality and normal morphology around 24 hours post-cooling with TY and TL thinners.

Table 2. Integrity of the acrosome around 24 hours post-refrigerating with the TY and TL diluents.

Table 3. Effect of the type of preservation of semen and diluent on reproductive parameters in primiparous rabbits artificially inseminated. TR = Cooling time. TY = Tris diluent + egg yolk. TL = Tris + skim milk. DG + = Positive pregnancy diagnosis. F = Fertility. GNT = Total born gulfs. TPC = Average litter size. PPC = Average litter weight.

4. Discussion

The motility, vitality, morphology and integrity of the plasma and acrosomal membranes are spermatic function parameters directly related to the fertilizing capacity, modified with long-term refrigeration. In this study, a decrease in progressive motility was observed from 12 and up to 24 hours after the refrigeration between diluents. Previous studies have shown a protective effect provided by the egg yolk on this parameter in several species (Crespilho et al., 2014; Alli et al., 2015; Sánchez et al., 2006). Regarding vitality and normal morphology, there were no significant differences (P> 0.05) neither in the conservation time nor between the diluents compared to the control. Rosato et al. (2006) reported a 65% vitality in refrigerated rabbit spermatozoa using a commercial diluent (Lepus) after 24 hours of refrigeration, showing a lower result as opposed to both diluents used in this study (TEY 79 ± 10.91% and TSM 75.57 ± 11.87%). This behavior is also observed for the normal spermatic morphology parameter. The results obtained in this study with TSM diluent are lower than those obtained with TEY diluent. Despite the widespread conventional use of skim milk as a component in semen conservation (Salamon and Maxwell, 2000), currently the action mechanism by which this component exerts its protective effect has not been fully clarified, although several mechanisms have been proposed (Bergeron and Manjunath, 2006). The results of acrosomal integrity in this study report that for 8 and 24 hours after refrigeration spermatozoa preserved with the TEY diluent presented higher integrity percentages in their acrosome (uncapacitated spermatozoa with an intact acrosome), showing 61.83% and 59.17% respectively compared to 56.17% (8 hours) and 50.5% (24 hours) corresponding to the TSM diluent. However, around the first 2 hours after refrigeration the behavior between diluents remained unchanged. These results are well above those obtained by Cresphilo et al. (2014) who reported 4.0% of intact acrosome in refrigerated bovine spermatozoa (5°C) around 24 hours, using a diluent based on Tris-egg yolk-fructose. The reproductive parameters of artificially inseminated females showed an independent behavior regarding the refrigeration time and type of diluent, except with the TEY, which exhibited a decrease in the percentage of fertility as the refrigeration time increased. Some factors related to the female subjects that intervene directly on the results are the state or physiological condition and age of reproductive life (Castellini and Lattaioli, 1999). In addition to factors directly related to the AI technique, such as the estrus induction method and the ovulation induction method (García and Rodríguez, 2002) and number of sperm per dose (Roca et al., 2000). Dixon and Anderson (2004) also mention that the female reproductive tract determines the environment in which the sperm would have to compete to fertilize the oocytes.

The favorable effects of egg yolk and skim milk, to counteract the alterations caused by cold shock, are amply proven in semen refrigeration in different domestic species. However, the complex mechanisms related to this protection are currently under investigation. Our results reaffirm the importance of adding egg yolk and skim milk to the conservation diluent in rabbit semen refrigeration, given the benefits in motility preservation, plasma and acrosomal membrane stabilization, which proved to be sufficient to achieve fertilization. Nevertheless, later studies should contemplate factors directly related to the parameters evaluated in this study. Factors such as race, reproductive seasonality, genetic line, physiological condition, as well as in the study of different components of the seminal conservation diluent. With the purpose of improving the maintenance and functionality of the spermatic cell, in addition to preserving the fertilization potential of the spermatozoa, under refrigerating or freezing protocols, with the aim of transmitting reproductive, productive, competitively desirable characteristics to future rabbit populations.

5. Conclusion

The tolerance of rabbit semen to conservation at 5°C up to 24 hours, measured in terms of maintenance of the basic parameters of spermatic quality (motility, vitality, morphology) and acrosomal integrity is influenced by the diluent used. However, the diluent with egg yolk in its composition shows a better behavior regarding the maintenance of the basic parameters of spermatic quality as opposed to the diluent based on skim milk. Nevertheless, the reproductive parameters of females artificially inseminated with refrigerated semen show an independent behavior regarding the conservation time and the diluent used. Based on the results obtained in this study, it is advisable to use egg yolk and skim milk as main components of the conservation diluent for the rabbit semen doses refrigerated at 24 hours to be used in artificial insemination.