Use of native chicken breeds (Gallus gallus domesticus) for the development of suitable methods of Cantabrian capercaillie (Tetrao urogallus cantabricus) semen cryopreservation

Abstract Background The Cantabrian capercaillie (Tetrao urogallus cantabricus) is critically endangered. This subspecies has the lowest genetic variability and it is in regression. It belongs to Phasianidae family; therefore, the domestic chicken (Gallus gallus domesticus) could be a good model for developing reproductive technologies for use in capercaillie populations with low availability of animals. Objectives In this study, we analyzed the response of capercaillie sperm to the freezing–thawing process for contributing to the development of a semen cryobank of Cantabrian capercaillie. Methods We used domestic chicken as the animal model in order to obtain the freezing protocol before applying on capercaillie. In the first experiment, two different extenders (EK and LR84) and different concentrations [4% and 6% dimethyl‐acetamide (DMA) v:v] of cryoprotectants were evaluated using in‐straw freezing method in domestic chickens. A pilot study in capercaillie males, using the same conditions evaluated in chicken, was performed. Results In chicken, we found that the LR84‐4% DMA media provided the best results for freezing semen. In capercaillie study, LR84 extender seemed to be the most appropriate diluent and 4% was the better dose of DMA cryoprotectant agent. Further, based on previous studies carried out in rooster samples, we also tested the glycerol (8% v/v) as a cryoprotectant for capercaillie semen cryopreservation. Conclusions Our results suggest that sperm from both domestic and wild species had a similar response to freezing–thawing processes. Mediterranean chickens may be used as a suitable model for developing sperm freezing protocols that can be extrapolated to threatened capercaillie populations. In addition, LR84 media with glycerol was the most efficient extender to freeze capercaillie sperm native.


cantabricus) inhabits deciduous forests of the Cantabrian Mountains of
Spain. This species faces a high risk of extinction in its native habitat, not only because of its peripheral location but also due to its small population size, low genetic diversity and low incoming gene flow (Alda et al., 2013). The primary causes of threat are multiple, such as the management of the habitat that involved increasing the density and transformation of forest edges, increasing predators, climate change and hunting. In 2019, the census carried out by the Ecological Transition Ministry of Spain (2019) reveals that there are only 292 specimens of Cantabrian capercaillie that survive in a 350 km 2 range of the ridge Cantabrian.
Conservation programmes and reproductive technologies can help to protect species from becoming extinct. Semen cryopreservation, coupled with artificial insemination, has become the main tool for threatened species in ex situ assisted reproduction. Also preserving their genetic diversity in genome resource banks is essential if the success of captive breeding programmes is to be guaranteed (Prieto et al., 2014).
Previous studies on the optimization of capercaillie semen cryopreservation protocols have been described in central European capercaillie populations (T. urogallus). Kowalczyk and Łukaszewicz (2015) reported two methods of freezing capercaillie semen using EK as extender and the permeant cryoprotectants 6% dimethyl-acetamide (DMA) for freezing in pellets and 6% of dimethyl-formamide (DMF) for freezing in straws. Their results showed that the straw method allows obtaining a higher survival rate and number of motile sperm, compared to pellets. However, there are no previous studies of cryoresistance of Cantabrian capercaillie semen. Reproductive techniques for use with a target species could be developed using phylogenetically related model species. Domestic chicken (Gallus gallus domesticus), a Phasianidae species as the capercalillie, might provide a good model for developing new reproductive technologies for use with threatened related wild species as capercaille, in which the number of available individuals is very scarce.
Based on the protocols developed in domestic chicken, we described, for the first time, the effect of different extenders and cryoprotectants in-straw freezing protocol of the Cantabrian capercaillie's semen. All semen collections in both chicken and capercaillie were performed using the dorsoabdominal massage technique (Burrows & Quinn, 1937;Łukaszewicz et al., 2011). In capercaillie males, semen was recovered by capillarity using a microhaematocrit tube (Brand GMBH + Co KG, Wertheim, Germany).

Assessment of sperm variables
The sperm concentration was estimated using a Neubauer chamber  equipped with a probe resistant to freezing. For thawing, the straws were warmed for 30 s in a water bath at 5 • C.

Experimental design
This study consisted of one experiment and a pilot study involving the cryopreservation of semen from native chicken breed and capercaillie, respectively. Experiment 1 involved the use of two different diluents with two different DMA concentrations in a Birchen Leonesa rooster.
The pilot study consisted of the use of capercaillie males, involving the same extenders with DMA as Experiment 1, and a Lake-Revie extender with glycerol as previously tested in native Mediterranean chicken breeds (Abouelezz et al., 2015).

Experiment 1: Cryopreservation of semen of the Birchen Leonesa rooster comparing two extenders and two different concentrations of DMA
This experiment studied the effect of two extenders, LR84 and EK, with two different concentrations of the DMA cryoprotectant (4% and 6%) on frozen-thawed rooster sperm variables.
Ten samples of five roosters (two for each animal) about 2 years old of the Birchen Leonesa were obtained. As the volume obtained from each male was not enough to perform the four treatments, half of the samples (n = 5) were immediately diluted 1:1 (v:v) using LR extender, and the other half of samples (n = 5) were diluted 1:1 (v:v) using EK extender. Then, each aliquot was refrigerated for 1 h at 5 • C. After the equilibrated period with the extender, a third volume of the extender with 12% DMA (final concentration 4% DMA) or 18% DMA (6% DMA final concentration) was added and equilibrated for 10 min at 5 • C.
After equilibration time, the samples were loaded into 0.25 ml French straws and frozen.
For this experiment, the comparison of each experimental condition was made analyzing the motility variables by CASA system, and the viability by fluorescence (PI/SYBR14) of the fresh and frozen/thawed rooster semen.

Pilot study: Cryopreservation of capercaillie semen comparing extenders previously studied in chicken
Under the hypothesis that the best conditions observed in roosters could be the same for capercaillie, we carried out this study using the same extenders and cryoprotectant agents previously evaluated in roosters.

2.8
Pilot study 2.1: Freezing semen of capercaillie using LR84 and EK extenders with 6% and 4% DMA as a cryoprotectant Semen samples of three capercaillie males were analyzed: one Cantabrian capercaillie male named URS1, and two European capercaillie males named URA1 and URA2. The samples obtained were divided into two aliquots. Both fractions were brought to a 1:1 (v/v) dilution, but each one with a different extender (EK or LR84), and refrigerated for 1 h at 5 • C. Then, DMA was added to leave a final 6% or 4% concentration and equilibrated for 10 min at 5 • C before freezing.
For this experiment, due to the scarce volume of the collected sample, the fresh sperm analysis consisted only the subjective motility and volume. For frozen/thawed samples, viability (PI/SYBR14), motility (CASA) and sperm DNA integrity (TUNEL) were analyzed. TA B L E 1 Rooster sperm quality variables (mean ± SE) of fresh and frozen-thawed samples with 4% of dimethyl-acetamide (DMA)

2.9
Pilot study 2.2: Freezing semen of capercaillie using LR84 extender and glycerol as a cryoprotectant The glycerol (GLY) as cryoprotectant was previously checked in native Mediterranean chicken samples (Abouelezz et al., 2015), returned higher sperm viability, intact acrosome and motility values. Thus, we tested the freezability of the Cantabrian capercaillie sperm with 8% GLY in the in-straw freezing method.
Samples from four of six Cantabrian capercaillie males (male identification: 31-C, 55-C, 61-C and 70-C) were obtained. Only the semen volume obtained from two of them was enough for freezing (31-C and 55-C). Both samples obtained were brought to a 1:1 (v/v) dilution in LR84 extender for 1 h at 5 • C. Then, the glycerol was added directly to diluted sperm samples to reach the final concentration of 8% (v/v).
For this experiment, the fresh sperm analysis consisting of the subjective motility, viability (eosin/nigrosin), sperm morphoabnormalities, acrosome status and DNA integrity were performed. For frozen/thawed samples, viability (PI/SYBR14 and E/N), motility and sperm DNA integrity (TUNEL) were analyzed.

Statistical analysis
The influence of the interaction semen media (LR84 and EK) × cryoprotectant concentration (DMA 4% and 6%) on sperm variables was analyzed by two-way analysis of variance (ANOVA), following the statistical model xijk = m + Ai + Bj + ABij + eijk, where xijk is the measured sperm variable, m is the overall mean of variable x, Ai is the effect of media (i = 1-2), Bj is the effect of DMA concentration (j = 1-2); ABij is the interaction between A and B and eijk is the residual (k = 1-10). A post hoc Newman-Keuls test was performed to compare differences between extenders. Comparisons between fresh and frozen-thawed sperm variables were made using a paired t test.
Data were expressed as means ± SE. All statistical calculations were made using Statistica software for Windows v.12 (StatSoft Inc., Tulsa, OK, USA).

RESULTS
In roosters, the values of all frozen/thawed sperm variables, except for motility score, linearity (LIN), straightness (STR) and wobble (WOB) were lower (p < 0.05) than those recorded for fresh sperm when the semen was cryopreserved using the 6% DMA (Table 1). In addition, the values of all frozen/thawed sperm variables except for LIN, WOB BCF were lower (p < 0.05) than those recorded for fresh sperm when the semen was cryopreserved using the 4% DMA (Table 1). Frozenthawed sperm viability, curvilinear velocity (VCL), straight-line velocity (VSL) and average path velocity (VAP) were greater (p < 0.05) using LR84-6%DMA extender than EK-6% DMA extender. There were no TA B L E 2 Capercaillie sperm quality variables of frozen-thawed samples using LR84 or EK extenders with 4% of dimethyl-acetamide (DMA) (Cantabrian capercaillie URS1) and 6% of DMA (European capercaillie URA1 and URA2) the percentage of total motility and progressive motility seems to be higher in samples frozen with LR84-4% DMA. However, the kinetic variables of motility (VCL, VSL, VAP, STR and ALH) were greater than using EK extender. In addition, the percentage of DNA fragmentation observed is higher in EK (4.5%) than LR84 extender (0%) ( Table 2, URS1 male).
The percentage of viable sperm after freezing was greater than those observed using DMA as a permeant cryoprotectant.

TA B L E 3
Cantabrian capercaillie sperm quality variables of frozen-thawed samples using LR84 extender with 8% of glycerol

DISCUSSION
In this study, we obtained and described the ejaculate of both  (Abouelezz et al., 2015), conversely with studies in red jungle fowl (Gallus gallus murghi) in which 6% DMA maintained higher postthaw quality and fertility (Rakha et al., 2017). Hence, it should be noted that the sperm of native Mediterranean breed of chicken are not very tolerant of DMA, and concentrations lower than 6% are recommended.
Despite suitable results have been found in semen European capercaillie frozen with 6% DMA (Kowalczyk et al., 2012), our results recommend that the lower DMA concentration should be used in future stud-ies with Cantabrian capercaillie. The method of freezing may also influence the results. The pellet method has been recommended when DMA is used as cryoprotectant (Tselutin et al., 1995). Indeed, first studies in capercaillie with DMA used the semen freezing in pellets (Kowalczyk et al., 2012). However, further studies by Kowalczyk and Łukaszewicz (2015) did not find differences between straw and pellet methods with 6% DMA as the cryoprotectant.
Previous findings (Abouelezz et al., 2015) showed that glycerol was more effective than DMA to cryopreserve sperm from native chicken breeds. The advantages of using glycerol as a cryoprotectant in chicken semen have also been established and recommend in the conservation of rare genetic resources (Thélie et al., 2019). In order to study if similar findings were found in capercaillie, we evaluated the effect of the  (Kowalczyk & Łukaszewicz, 2015). Our data showed that frozen-thawed sperm quality variables of the 31-C male were better than the 55-C male. In this regard, it is well known that the sperm ability to be cryopreserved can vary between individuals. Moreover, other earlier studies in European capercaillie indicated that the quality of ejaculates depends on individual male properties and their age but not the way of male management (Kowalczyk & Łukaszewicz, 2015).
The initial quality of fresh semen sample plays a relevant role in freezing-thawing suitability. The Cantabrian capercaillie volumes were lower in comparison with previous studies in European capercaillie (Ciereszko et al., 2011;Łukaszewicz et al., 2011). Also, sperm concentrations obtained in Cantabrian capercaillie were lower than those found in European capercaillie by Kowalczyk et al. (2012) and Kowalczyk and Łukaszewicz (2015). Perhaps, the volumes and concentration of the Cantabrian capercaillie ejaculated are naturally less than what is seen in European capercaillie located in central Europe, or maybe the bottleneck of the Cantabrian population determines a strong inbreeding that finally affects the semen quality.
The high DNA damage levels in fresh Cantabrian capercaillie semen samples of the pilot study 2.2 are remarkable (from 19% in 31-C to 28% in 55-C). Regarding this, a link has been reported between sperm DNA damage and inbreeding in ungulates (Ruiz-Lopez et al., 2010). Our data also showed that semen quality from European capercaillie males of the zoological garden also showed low quantitative characteristics, which may reveal inbreeding of this captive population.
Conversely, the quality of the fresh samples for Cantabrian capercaillie (URS1 male of the pilot study 2.1 and all males of the pilot study 2.2) was similar to those observed in European capercaillie in previous studies. While we observed 55%-80% of total motility in fresh semen, Kowalczyk and Łukaszewicz (2015) observed 82% average motility and Ciereszko et al. (2011) reported 71% motility after 2 h of short-term storage in EK extender. Fresh sperm viability, ranging between 77% and 85% in Cantabrian capercaillie, was similar to European capercaillie semen (Kowalczyk & Łukaszewicz, 2015;Kowalczyk et al., 2012;Łukaszewicz et al., 2011).

CONCLUSION
In conclusion, our results suggest that sperm from both domestic and wild species had a similar response to freezing-thawing processes.
Mediterranean chickens may be used as a suitable model for developing sperm freezing protocols that can be extrapolated to threatened capercaillie populations. In addition, LR84 media with glycerol was the most efficient extender to freeze capercaillie sperm native. Although the sample size of this study is small and it is necessary to carry out more studies to obtain consistent and comparable results, it should be noted that this is the first description of semen freezing in Cantabrian capercaillie for the future development of a germplasm bank in this subspecies.