Background
Establishing a genome resource bank, along with assisted breeding techniques, has the potential to preserve genetic diversity of many endangered species managed ex situ [
1]. Frozen semen and embryos can be used to minimize inbreeding and genetic drift in small populations. Moreover, the subsequent risk of expression of unfavourable or even lethal genetic traits, as well as the loss of advantageous characteristics through unnatural selection, can be lowered [
2]. We have reported the potential application of semen cryopreservation in a program of gamete and embryo banking, designed to restore genetic diversity in small and isolated populations of wild ruminants, such as the European mouflon (Ovis gmelini musimon) and endangered gazelle (Gazella dama mhorr), through both in vivo and in vitro reproductive techniques [
3‐
5].
In the case of birds, it is not possible to cryopreserve embryos or oocytes, mainly because of their large size, the high lipid content, and the polar organization [
6]. Therefore, the most feasible method for ex situ management of genetic resources in birds is semen cryopreservation. The feasibility of the construction and utilization of an avian semen cryobank for the ex situ management of endangered pheasants and of rare domestic lines and breeds of the species
Gallus gallus has recently been reported [
7,
8]. Artificial insemination with frozen and thawed semen has also been used to produce chicks in non-domestic bird species, such as the American kestrel [
9], the Sandhill crane [
10], the Canadian goose [
11], and the Budgerigar [
12]. It is noteworthy that researchers have promptly responded to the well known catastrophic collapse in the population of the Indian white-backed vulture (
Gyps bengalensis) by studying its basic spermatology and developing a protocol for semen cryopreservation in order to conserve the specie through captive breeding and assisted reproduction [
13].
Endangered bird conservation efforts can thus rely on the application of assisted reproduction techniques and the integration of long-term intensive demographic and genetic monitoring with the parallel creation of a semen cryobank of the local threatened population would guarantee a "life insurance" for the specie.
In Sardinia, Italy, out of three large vulture species present until the mid-twentieth century (
Aegypius monachus,
Gypaetus barbatus and
Gyps fulvus), only the Griffon vulture (
Gyps fulvus) has survived until today. Distributed over the whole island up to the late 1940s with an estimated population of 800 – 1200 individuals, the population of Griffon vultures in Sardinia dropped very rapidly after the Second World War, mainly in relation to the use of poisoned baits, outlawed in 1977. In 1975 the estimated population counted only 100 – 140 Griffon vultures [
14]. In order to save the last Griffon vulture native population in Italy from extinction, several conservation projects focused on food supplementing and creation of new protected areas have been carried out during the last 30 years. Despite these efforts, different poisoning actions in 1997/98 and the further loss of 30–35 individuals in 2006/07 reduced the population to the present 60–65 individuals and 21–22 breeding pairs, concentrated by more than 90% in the north-west of Sardinia. This situation begs for the development of additional conservation strategies.
The aim of our study was to test the feasibility of a programme of semen collection and cryopreservation in Griffon vultures. Semen collection efficiency, ejaculate traits and post-thaw semen quality, as evaluated by its in vitro survival rates, DNA damage and ATP concentration, were determined. Considering that the freezability of avian spermatozoa appears to be very different between species [
15], even when showing very similar morphological shapes and ultrastructures [
16,
17], this information is fundamental in order to start establishing a semen cryobank of Griffon vultures. We took advantage of the presence of a local wildlife rescue centre where Griffon vultures are temporary recovered for sanitary problems and where a few wild-caught individuals originating from the natural population are permanently held in captivity because of unrecoverable traumas.
Discussion
This study reports original data on the basic reproductive parameters of the Griffon vulture, in terms of semen characteristics and ability to survive freezing and thawing procedures. Furthermore, a method to determine intracellular ATP concentration in as little as 20.000 total spermatozoa was used, allowing to perform this assay even in very small semen samples and thus to provide useful information about the energetic status of the sperm cells. Although the results reported here are based on a limited number of individuals, a common problem when dealing with endangered species, our data are important for the future development of genetic resource banks for this and related species. Besides, as part of this endeavour, it is essential to take the opportunity to learn about the basic reproductive biology of the species to be preserved.
The present study demonstrates that the abdominal massage method for semen collection can be used to induce ejaculation in the Griffon vulture, but the efficiency with which the vultures ejaculated (27.9%) was lower compared to the rates obtained in other birds, such as the Indian white-backed vulture [84.6%; [
13]], the American kestrel [74%; [
26]], the sandhill crane [86%; [
27]], and the blue rock pigeon [90%; [
28]], using the same method. Besides, we obtained ejaculates of rather small volumes (12.5 ± 9.1 μl) and with a high variability in sperm concentration (28.4 ± 30.9 spermatozoa × 10
6/ml). In the Indian white-backed vulture (
Gyps bengalensis), which is the closest specie we could refer to, ejaculate mean volume was 370 ± 360 μl, while average sperm concentration was 58.4 ± 33.2 spermatozoa × 10
6/ml [
13]. Total sperm output was therefore considerably lower in Griffon vultures used in the current study.
Since no female vulture was present in the wildlife rescue centre during this study, the use of visual contact with females in the case of Umapathy's study may explain the difference [
29,
30]. Researches on the role of behavioural stimuli in regulating the generation of mature gametes in birds are lacking. Cecil and Bakst [
31] failed to find a positive influence of hen visual contact on ejaculate volume, concentration, and total spermatozoa output in male breeder turkeys. On the other hand, a more recent study reported that the confrontation with a female conspecific for 60 min positively affected plasma testosterone concentrations in male European starlings (
Sturnus vulgaris) [
30]. This finding agrees with the results of earlier studies [
32,
33]. It is generally recognized that testosterone plasma concentrations are positively related to sperm production since the hormone is needed for normal spermatogenesis and it is closely associated with testes size [for review see: [
34]]. Differences in the behavioural response to human handling may also account for the low semen collection efficiency reported in this study for wild-caught Griffon vultures. Several methods have been used to obtain semen from birds: (1) killing and dissection of birds to remove sperm from the reproductive tract or testes [
35]; (2) abdominal massage, which was developed by poultry biologist to obtain semen for artificial insemination [
36,
37]; (3) the application of a "sperm collector" to the cloaca [
38]; (4) cooperative semen collection, in which imprinted birds voluntarily copulate on special devices in response to behavioural stimuli [
39]; (5) use of a female dummy [
40].
All of these techniques have limitations. The principal side effects of the abdominal massage method is that it is a stressful procedure, since it involves catching and handling of the birds, and that it stimulates urination, with consequent contamination of the ejaculates [
10,
39]. We had to discard the 62.2% of total ejaculates because of urine/faecal contamination or low viability, and this problem has already been described in other birds of prey [
13,
15].
Nevertheless we choose this method because it was the less invasive for not imprinted birds and it had been already tested on vultures [
13]. Better results in terms of ejaculate induction in response to the stimulation and sperm output may be eventually obtained with birds raised in captivity and kept in visual contact with a female conspecific.
Considering that the Griffon vulture population in Sardinia dropped from 800 – 1200 to 100–140 individuals very rapidly and that there are no reports about the immigration of individuals from other colonies [
14], we can also speculate that inbreeding depression consequent to isolation and small population size may account for the low semen output obtained in the current study. It is known that inbreeding reduces male reproductive function and semen quality [
41,
42]. In the endangered gazelle,
Gazella cuvieri, the individual's coefficient of inbreeding correlated negatively with many parameters of semen quality, including ejaculate volume [
43]. Unfortunately, information on genetic variability in the Sardinian Griffon vulture is lacking.
In order to assess Griffon vulture semen cryotolerance and eventual differences in post-thawing semen quality among the four individuals, in vitro viability, DNA integrity and ATP concentration in frozen/thawed spermatozoa were evaluated. Our data showed that spermatozoa viability after thawing did not differ between the four individuals, but it decreased significantly compared to fresh semen (p < 0.05).
Avian semen is particularly susceptible to freezing damage [
6,
44]. The high incidence of morphological disruption and ultrastructural abnormalities after cryopreservation have been related to its filiform shape and long tail which makes avian semen more susceptible to injury from mechanical manipulation [
45,
46]. Furthermore, poultry spermatozoa are more susceptible to osmotic stress that bovine spermatozoa [
47] and their function is adversely impacted by excessive dilution [
48]. Studies in chickens and turkeys have shown changes in many functions of spermatozoa, including viability, motility and ATP concentration after freezing [reviewed by refs. [
44,
49]].
In our study we reported that Griffon vulture spermatozoa can tolerate freezing and thawing procedure and are able to survive during 4 hrs in vitro culture. Noteworthy, after freezing/thawing spermatozoa collected from B maintained a higher viability during in vitro culture compared to spermatozoa from the other vultures.
Within-individual variation in the susceptibility of sperm cells to cryoinjury has been described in many different species [for review see: [
50]]. Two biophysical traits of bird semen, i.e. resistance of spermatozoa to osmotic stress and membrane fluidity, have been reported to be possibly related to the differences between species and individual in the ability of spermatozoa to withstand the freezing-thawing process [
51].
DNA fragmentation after freezing and thawing did non differ in the 4 vultures as evaluated by the comet assay method. The lack of repair mechanisms in sperm makes DNA damage irreversible. Griffon vultures frozen/thawed spermatozoa do not appear to be particularly susceptible to DNA fragmentation during cryopreservation since the mean scored parameters are similar to those recorded in turkey spermatozoa kept 2 hrs at 4–7°C [
25]. Since cryopreservation may affect sperm function at many levels, in order to better assess Griffon vulture spermatozoa cryotolerance, adenosine triphosphate (ATP) intracellular concentrations were determined before and after cryopreservation. Individual differences in nucleotide levels in fresh semen were observed. This result is consistent with data reported for turkey spermatozoa, where most males segregate into characteristically high, average or low ATP categories [
52]. Similarly a wide range of variability in ATP concentrations was described for fresh individual rooster semen [
44,
52]. Energy metabolism is a key factor supporting sperm function. Sustaining sperm motility and active protein modifications such as phosphorylation could be the reason why sperm require exceptionally more ATP than other cells [
53]. The existence of different sperm mobility phenotypes in roosters has been already pointed out [
54] and subsequently it has been related to differences in mitochondrial function [
55]. In this study, an higher ATP concentration in fresh semen was followed by a longer survival in vitro after cryopreservation. Furthermore, its values correlated positively with sperm viability both before and after cryopreservation. It is generally accepted that assessment of the quality of fresh semen helps to predict the suitability of spermatozoa to withstand freezing and thawing. It would be interesting to investigate whether ATP intracellular concentration could be used as a tool to select the most suitable donor for semen cryopreservation in this and related species.
The extent of ATP loss in Griffon vulture spermatozoa was severe after cryopreservation. This result agrees with data reported for rooster [
44] and crane spermatozoa (
Grus leucogeranus) [
56]. Given the apparent sensitivity of avian sperm mitochondria to the freeze/thaw process, it is not surprising that ATP production would be compromised after cryopreservation [
44]. As a cause for low fertility of cryopreserved semen after artificial insemination, mitochondrial damage caused by freezing and thawing has been suggested [
57,
58]. Studies with isolated mitochondria proved that freezing and thawing largely impairs their bioenergetic functions [
59]. These reports suggest that mitochondrial damage during cryopreservation is a major cause of low post thaw semen quality, but it is not adequately diagnosed with routine laboratory methods.
In conclusion, this study provides original information on Griffon vulture semen characteristic and cryotolerance, and points out the many difficulties that still prevent the widespread use of semen cryopreservation in the conservation of genetic resources in wild birds. Other techniques, such as the long-term preservation of primordial germ cells [
60], the cryopreservation and transplantation of testicular tissue [
61], and interspecies germ cell transplantation [
62], are being studied but are not yet considered to be fully appropriate to the ex situ management of genetic resources. Semen cryopreservation may be considered as a useful tool in the management of captive populations of Griffon vultures, but further studies aimed to optimize semen collection efficiency in wild-caught birds and to test cryopreserved spermatozoa fertilizing potential are needed. Furthermore, accurate genetic analysis are needed to assess the genetic variability of the Griffon vulture Sardinian population.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
MM performed the semen collection and analysis, and helped drafting the manuscript. FB participated in the design of the study, carried out experimental analysis, performed the statistical analysis and drafted the manuscript. GGL participated in the design of the study and performed the statistical analysis. VS performed the DNA analysis for the individuation of the sex of the animals and participated in semen analysis. VP helped in performing the DNA and ATP analyses. ML, SS and AR helped in sample collection and in drafting of the manuscript and participated in the design of the study. AZ and CC performed the ATP analysis. MM conceptualised the idea, helped in drafting the manuscript and revising it critically for important intellectual content. SN conceived of the study, participated in its design and coordination and helped to draft the manuscript. All co-authors provided inputs during final manuscript preparation. All authors read and approved the final manuscript.