Advances in sperm cryopreservation of samples colleted by vagina artificial and electroejaculation from Blanca-Celtibérica goat breed

Resumen

Blanca-Celtibérica goat is an endangered autochthonous breed from Spain. To prevent the disappearance of these autochthonous breeds and in order to maintain the genetic diversity, the Food and Agriculture Organization of the United Nations (FAO) recommends the preservation of these breeds by in situ and ex situ conservation programs (FAO, 2010). The creation of Genetic Resource Banks (GRB) is a measure of ex situ conservation and allows us the storage of semen, oocytes and embryos indefinitely, being an essential tool in order to preserve the genetic diversity of species. The most widespread application in the development of GRB has been the semen collection and its freezing. This requires the knowledge of reproductive physiology as well as the suitable assisted reproductive technologies for each species. In the case of the Blanca-Celtibérica goat breed, the geographical location in addition to extensive production systems and the lack of buck center make semen collection by routine techniques not feasible. Thus, when animals can not be trained to semen collection by artificial vagina, there are other alternatives such as the electroejaculation, being possible to obtain semen of males living in the countryside. Besides the semen collection, the conservation of semen samples is also a key aspect. Up to date, although several studies have been carried out on sperm cryopreservation in caprine, most of them have used samples collected by artificial vagina. However, it is known that the semen collection method influences on the sperm production and composition of seminal plasma (Marco-Jiménez et al., 2005; Marco-Jiménez et al., 2008) as well as the cryoresistance of sperm samples (Álvarez et al., 2012). Another problem during the buck semen cryopreservation is the low seminal quality found after thawing when freezing extenders based on egg yolk or skim milk are used. Some components of egg yolk and skim milk have negative interactions with seminal plasma as a result of the production of toxic compounds to the spermatozoa. This effect could be more pronounced on sperm samples collected by electroejaculation since the volume of seminal plasma is higher than in samples obtained by artificial vagina. With this background, the general aim of this Doctoral Thesis has been to develop an optimal method in order to collect and cryopreserve buck semen of Blanca-Celtibérica breed which allow us to storage seminal doses to create a Germoplasm Bank. In the first Chapter of this Doctoral Thesis, it was evaluated the effect of method of semen collection, artificial vagina and electroejaculation, on sperm quality at thawing in Blanca-Celtibérica buck, comparing the results obtained in other close-related small ruminant species, sheep, which have more available information regarding to cryopreservation (Marco-Jiménez et al., 2005; Marco-Jiménez et al.2008; Álvarez et al., 2012). The goal was to establish a starting point to cryopreserve semen of Blanca-Celtibérica goat breed using a standard freezing protocol performed for small ruminants, and two collection methods, artificial vagina as usual collection method for small ruminant, and electroejaculation as alternative method. In both species, the sperm samples collected by electroejaculation had higher volume and lower concentration and no differences were observed for sperm motility for ejaculates obtained by both collection methods. However, the collection method and species influenced on the resistance of sperm samples to freezing. Thus, after thawing, the sperm motility parameters were lower in bucks than rams, being the lowest values those for samples collected by electroejaculation in bucks. Moreover, for this species and collection method were showed lower values of viability and active mitochondria and higher DNA damage than those collected by artificial vagina and ovine samples collected by both methods. In the second Chapter of this Thesis, the effect of ejaculate collection method (artificial vagina or electroejaculation) and season of collection (breeding season or non-breeding season) on thawed sperm quality were studied in Blanca-Celtibérica goat. In addition, it was evaluated the influence of seminal plasma removal by centrifugation before freezing and the use of different freezing extenders, two commercial, Biladyl® (based on egg yolk) and Andromed® (based on soy lecithin) and a non-commercial extender based on skim milk. Finally, interactions between studied factors were also analysed. Samples obtained by electroejaculation showed lower sperm quality at thawing than those collected by artificial vagina. Moreover, the season of collection influenced on the sperm quality of thawed samples, being more suitable the sperm collection during breeding season irrespective of the collection method. The removal or not of seminal plasma before freezing by centrifugation had not any effect on sperm quality at thawing regardless of collection method and extender used. However, it was observed a beneficial effect of seminal plasma removal when the semen was collected during non-breeding season. Otherwise, commercial extenders based on egg yolk and soybean (Biladyl® and Andromed®, respectively) provided good values of seminal quality at thawing in relation to skim milk, being even higher for viability when Biladyl® was used. Due to poor sperm quality at thawing showed in the previous experiments for samples collected by electroejaculation in relation to artificial vagina, it was necessary to carry out modifications in the freezing extender and procedure in order to improve the sperm quality after cryopreservation. Therefore, it was proposed the next Chapter of this Doctoral Thesis. In the third Chapter of this Thesis, the effect of removal of the seminal plasma by sperm selection using single layer centrifugation (SLC), before freezing and after thawing, on sperm quality at thawing was researched. Thawed sperm samples selected by SLC before freezing showed lower sperm quality than samples selected after thawing or not selected. The best sperm quality was observed in those samples selected after thawing. Moreover, these sperm samples had higher percentage of DNA integrity. However, the sperm recovery after SLC was very low for samples selected at thawing in relation to those selected before freezing. Although high sperm quality was obtained when SLC was performed after thawing, the total number of recovered spermatozoa was lower than for samples selected before freezing and so, this technique is not suitable from a technical standpoint. Therefore, a fourth experiment was proposed. In the fourth Chapter of this Thesis were modified some aspects of freezing extender as well as other related to cryopreservation protocol in order to obtain high quality sperm samples. In this way, the effect of egg yolk concentration (0, 1.5, 10 y 20%) contained in the freezing extender was studied. It was also evaluated the effect of cooling rate (decrease of temperature from 30 ºC to 5 ºC in 90 minutes (slow rate) or 10 minutes (fast rate)), the effect of temperature of glycerol addition (30 ºC or 5 ºC) and the effect of equilibration time at 5 ºC (0, 1, 2 or 3 h). Extenders containing 10 and 20% of egg yolk provided higher sperm quality after thawing than those with 0 and 1.5 %, showing 20% of egg yolk similar values than control samples (those collected by artificial vagina and frozen with a standard protocol; 20% egg yolk, slow cooling rate, addition of glycerol at 5 ºC and 2h of equilibration), except for motility parameters. Moreover, slow cooling rates (90 min) had higher sperm quality than the fast ones (10 min) and no differences were observed in relation to control samples. Otherwise, no differences were found between temperatures of glycerol addition and longer equilibration periods at 5 ºC (3 hours) provided better results at thawing than shorter periods (1 hour) or no equilibration time. Finally, a heterologous in vitro fertilization test was performed using samples cryopreserved with the best protocol observed for samples collected by electroejaculation (20% egg yolk, slow cooling rate and equilibration for 3 h) and the results were compared to sperm samples obtained by artificial vagina and frozen by standard protocol (control samples). The fertility of samples collected by electroejaculation and frozen by protocol which better results provided was slightly higher than for those obtained by artificial vagina and frozen by the standard protocol. From the results obtained in the essays presented in this Doctoral Thesis we can conclude that the thawed sperm quality in Blanca-Celtibérica bucks is influenced by several factors such as the collection method, the season of the year, the freezing extenders, their composition and the addition procedure, the cooling rate and the time which sperm samples are maintained at subphysiological temperatures. Hence, it is recommended to carry out the semen collection during the breeding season, to remove the seminal plasma if collection is performed during non-breeding season, and to freeze in extenders based on egg yolk such as Biladyl®, irrespective of the collection method. Moreover, higher thawed sperm quality for samples collected by electroejaculation were obtained when extenders with 20% egg yolk, slow cooling rates (from 30 ºC to 5 ºC in 90 minutes) and equilibration period for 3 hours were used. The temperature of glycerol addition can be performed at 5 ºC or 30 ºC. Finally, the sperm quality at thawing is improved when sperm samples collected by electroejaculation are selected by SLC after thawing, but not when samples are selected before freezing.