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BOR - Papers in Press, published online ahead of print July 20, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.043430
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BIOLOGY OF REPRODUCTION 73, 1039–1045 (2005)
DOI: 10.1095/biolreprod.105.043430
© 2005 by the Society for the Study of Reproduction, Inc.

Semen Characteristics of the Captive Indian White-Backed Vulture (Gyps bengalensis)1

Govindhaswamy Umapathy , Sadanand Sontakke , Anuradha Reddy , Shakeel Ahmed , and S. Shivaji 2 

Centre for Cellular and Molecular Biology, Hyderabad 500 007, India


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The present paper describes, to our knowledge for the first time, the successful collection and evaluation of semen from the Indian white-backed vulture (Gyps bengalensis), a critically endangered bird. Over a period of 2 yr, semen was collected using the manual massage method and evaluated for semen volume, semen pH, sperm concentration, percentage normal/abnormal spermatozoa, and percentage motile spermatozoa. It appears that the concentration of spermatozoa and percentage motile spermatozoa in the Indian white-backed vultures are low compared to those in other birds. Tyrode medium supplemented with albumin, lactate, and pyruvate (TALP) proved to be the best semen extender compared to two others (Beltsville Poultry Semen Extender and Lake diluent). Furthermore, TALP with 20% egg yolk and supplemented with 8% dimethyl sulfoxide maintained 50% of the initial percentage of motile spermatozoa following cryopreservation and thawing. A computer-aided semen analysis indicated that the spermatozoa of the Indian white-backed vulture are extremely active and swim in linear trajectories for up to 5 h following dilution in TALP. The trajectories were linear with time, but we noticed a decrease in the velocity parameters (average path velocity, curvilinear velocity, and progressive velocity). Thus, the present study provides baseline data on semen characteristics of the highly endangered Indian white-backed vulture, and these data could be of immense importance to reproductive and conservation biologists attempting to breed these animals in captivity, which to date has not been achieved.

gamete biology, sperm, sperm motility and transport


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the early 20th century, the Indian white-backed vulture (Gyps bengalensis) was abundant, and its distribution extended from the Thai-Malay peninsula and Indochina in the east to the Indus River, running through Pakistan, in the west [1]. However, a recent survey in India indicated a catastrophic collapse in the population of G. bengalensis, which had been totally wiped out from 17 key areas in that country [2]. This collapse was attributed to the toxic and detrimental effects of pesticides (which because of indiscriminate use ultimately accumulate in animal corpses on which the vultures feed), to viral disease, and more recently, to residues of a veterinary drug, diclofenac, in dead cattle [2–5]. Therefore, an immediate need exists to conserve the species either by captive breeding or by assisted reproduction. Captive breeding in birds of prey is not always successful because of stress-related behavioral changes [6]. Therefore, assisted reproduction may be the method of choice, but before resorting to assisted reproduction by artificial insemination, the basic spermatology of the Indian white-backed vulture needs to be understood [7, 8].

In the present study, semen was collected from the Indian white-backed vulture and evaluated for semen characteristics, such as semen volume, semen pH, spermatozoa concentration, percentage motile spermatozoa, and percentage normal/abnormal spermatozoa. In addition, motility kinetics of fresh and postcryopreserved spermatozoa also was studied using a computer-aided semen analysis (CASA) system. To the best of our knowledge, the present report is the first regarding the semen characteristics of the Indian white-backed vulture.


   MATERIAL AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Four Indian white-backed vultures from the Nehru Zoological Park, Hyderabad, India, were sexed and housed separately in outdoor pens under natural photoperiod. The vultures were in visual contact of females in the adjacent cages. The age of the vultures ranged from 17 to 25 yr, and weight ranged from 4.2 to 5.7 kg. The birds were fed with beef for 6 days in a week, and water was available ad libitum. All animal experiments were performed in accordance with the guidelines of the Central Zoo Authority of India, Ministry of Environment and Forests, Government of India. In addition, the work was approved by the Institutional Animal Ethics Committee of the Centre for Cellular and Molecular Biology, Hyderabad.

Semen Collection

Semen from the Indian white-backed vulture was collected using the massage method [9–13], which was modified because of the huge size and aggressive nature of the bird. Before semen collection, the bird was herded into a corner of the aviary and captured using a sweep-net (diameter, 45 cm). The captured vulture then was manually restrained, and the head and beak were covered with a cotton sock that could be easily slipped over the head and bill and fastened around the back of the head. A team of five people was required for the collection of semen from the vulture. Three persons were required to hold the limbs, the head, and the wings, respectively. The fourth person massaged the bird's back and used the other hand to simultaneously massage the bird from the midabdomen to the vent. The massage was done for 1 or 2 min. Following the massage, the bird may respond by everting its cloaca. The cloaca was then grasped by the fourth person using the thumb and index finger and the semen collected by the fifth person using a glass funnel [14]. The white-backed vulture breeds between September and February [1]. Therefore, semen collection attempts were made during this season, and ejaculates were obtained during the months of October to December. Ejaculates were not obtained if the vultures were massaged before or after the breeding season.

Semen Evaluation

Immediately after semen collection, the neat ejaculate was evaluated for semen volume, pH, sperm concentration, percentage motile spermatozoa, and percentage normal/abnormal spermatozoa. Ejaculate volume was estimated by aspirating the semen into a calibrated positive-displacement pipette and the pH by using pH indicator strips (Qualigens Fine Chemicals, Glaxo India Ltd., Mumbai, India), and the concentration of sperm using a Makler chamber as described by Sontakke et al. [14]. The percentage motile spermatozoa was determined using a phase-contrast microscope at 400x magnification, and a minimum of four separate fields were examined. For sperm morphology studies, 2 µl of neat semen was fixed in 100 µl of 0.5% gluteraldehyde, smeared on a glass slide, and observed under the microscope (400x). Approximately 100 spermatozoa per ejaculate were analyzed for sperm pleiomorphism.

Optimization of Semen Extender

Freshly ejaculated and uncontaminated semen samples were pooled and diluted (1:1 semen:extender) into various semen extenders, such as Tyrode medium supplemented with albumin, lactate, and pyruvate (TALP) [14], Beltsville Poultry Semen Extender (BPSE) [15], and Lake diluent (100 mM sodium glutamate, 44 mM fructose, 4 mM magnesium acetate, 51 mM potassium acetate, and 30 µM polyvinylpyrrolidone; 300 mOsm) [16] and then evaluated for the sustenance of motility of vulture spermatozoa. The diluted semen samples were maintained at 37°C in a CO2 incubator, which was continuously flushed with 95% air and 5% CO2. In preliminary experiments, it was observed that storage of diluted semen either at 5 or 25°C led to cessation of motility within 2 h. The percentage of motile spermatozoa was determined every hour using a CASA system (HTM IVOS 10; Hamilton Throne Research, Inc., Danvars, MA) as reported previously [17–22]. Significant differences between the extenders was calculated by the Mann-Whitney U-test. The experiment was repeated at least six times.

Effect of Dilution on Sperm Motility

Freshly ejaculated and uncontaminated semen samples were pooled; diluted in TALP in the ratios (semen:TALP) of 1:1, 1:2, 1:5, and 1:10; and incubated at 37°C in a CO2 incubator. The percentage of motile spermatozoa was determined every hour using the CASA system. The experiment was repeated five times.

CASA Analysis

To determine the motility parameters of Indian white-backed vulture spermatozoa, uncontaminated vulture semen was diluted in TALP [14, 17, 18] and maintained at 37°C in a CO2 incubator as above. After 15 min of dilution, 12 µl of sperm suspension were transferred to a prewarmed slide chamber (depth, 130 µm) [14, 18] and analyzed on an hourly basis using CASA. Various sperm motility parameters, such as average path velocity (VAP), progressive velocity (VSL), curvilinear velocity (VCL), beat cross frequency (BCF), straightness of track (STR), amplitude of lateral head displacement (ALH), and linearity of track (LIN) of the spermatozoa were determined as reported previously [14, 18, 21–24]. The incubation was continued until the spermatozoa were immotile.

Cryopreservation of Semen

Uncontaminated semen samples with more than 60% motile spermatozoa were pooled and then diluted 1:1 in TALP at 37°C, and aliquots of 750 µl were transferred to 1.8-ml screw-capped cryovials (Nalge International, Denmark) containing an equal volume of TALP supplemented with the cryoprotectants dimethyl sulfoxide (4% and 8%) or glycerol (4% and 8%) and 20% egg yolk. The TALP and 20% egg yolk without the cryoprotectant served as the control. The cryovials were then subjected to freezing in a programmable cryogenic unit (Consarctic, Gottingen, Germany) according to the following regime: temperature drop of 24 to 4°C at 1°C/min, then a drop of 4 to –85°C at 6°C/min, and then plunging the cryovials directly into liquid nitrogen [14]. Forty-eight hours later, the samples were thawed in a water bath at 37°C for 1 to 2 min and evaluated for percentage postthaw motile spermatozoa by using CASA. The experiment was repeated four times.

Statistical Analysis

Data are presented as the mean ± SD. For comparison of values between semen parameters, nonparametric Mann-Whitney U-test was used. Significant changes in the time-dependent motility parameters were evaluated by applying the Student t-test. Spearman rank correlation was used to test correlation among the semen characteristics. Statistical analysis was performed using the statistical software program SPSS (Ver 11.1 for Windows; SPSS, Inc.), and a probability of P ≤ 0.05 was considered to be the minimum level of significance.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Semen Collection and Semen Characteristics of White-Backed Vulture

A total of 78 attempts were made to collect semen from four males during the months of October to December of the years 2003 and 2004 using the massage method. Of 78 attempts, 66 (84.6%) ejaculates were obtained, and the characteristics of the ejaculates are summarized in Table 1. The color of semen varied from pale gray to milky white. From the mean values, it would appear that the uncontaminated semen of white-backed vulture is neutral in pH and has more than 50 x 106 sperm per milliliter of ejaculate. In addition, more than 50% exhibit normal morphology, and approximately 50% of the spermatozoa are motile (Table 1). A significant positive correlation was observed between sperm concentration and sperm motility (rs = 0.74, P < 0.001) as well as sperm concentration and semen pH (rs = 0.35, P < 0.05) using Spearman rank correlation. Evaluation of the semen characteristics of the four vultures separately (Fig. 1) indicated that vulture 577, with high sperm concentration, motility, and semen volume, could be the best individual for breeding, though attempts should be made to breed all four individuals to maintain maximum genetic diversity.


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  		TABLE 1. Characteristics of the uncontaminated and contaminated semen of the Indian white-backed vulture.a



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  		FIG. 1. Box plots (using SPSS 11.1) showing individual variation in semen volume (A, a), percentage motile spermatozoa (B, b), sperm concentration (C, c), percentage abnormal spermatozoa (D, d), and pH of semen (E, e) in contaminated (A–E; n = 6, 4, 4, and 14 for vultures 574, 575, 576, and 577, respectively) and uncontaminated (a–e; n = 7, 9, 12, and 12 for vultures 574, 575, 576, and 577, respectively) semen of Indian white-backed vultures. Different alphabets inside the box indicate that the values are significantly different (Mann-Whitney test, P < 0.05). An open circle indicates outliers, and an asterisk indicates extremes. An outlier represents a value between 1.5 and 3.0 box lengths from the upper or lower edge of the box, whereas extremes represent a value more than 3.0 box lengths from the upper or lower edge of the box

Urine/Fecal Contamination in the Semen Ejaculates

In the present study, 26 of the 66 ejaculates were contaminated (39.4%) either with urine or fecal matter (Table 1). Comparison of the semen characteristics of the contaminated and uncontaminated semen indicated that mean volume of the contaminated semen was significantly high (0.85 ml; Mann-Whitney U-test, P = 0.005) and had lower sperm concentration (31.3 million per milliliter; Mann-Whitney U-test, P = 0.007). However, other semen characteristics, such as the percentage motile spermatozoa, pH of semen, and percentage abnormal spermatozoa, did not show any significant difference (Mann-Whitney U-test; P = 0.28, P = 0.16, and P = 0.49, respectively) between the contaminated and uncontaminated ejaculates.

Optimization of Semen Extenders and Dilution

We found TALP to be the best among the three extenders for dilution of vulture semen. In TALP, more than 50% of the vulture spermatozoa were motile by 2 h of incubation compared to 22% in BPSE and 10% in Lake diluent (Fig. 2). Therefore, semen was diluted in TALP in the ratio of 1:1, 1:2, 1:5, and 1:10 to identify the optimal dilution for short-term storage. Semen diluted 1:1 with TALP maintained significantly higher motility (Mann-Whitney U-test, P < 0.05) up to 2 h (50%) compared to 1:2, 1:5, and 1:10 dilution (Fig. 3).



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  		FIG. 2. Effect of semen extenders on the motility of Indian white-backed vulture spermatozoa (black triangle, TALP; black square, BPSE; black circle, Lake diluent). Uncontaminated semen was diluted with the respective extender (1:1), incubated at 37°C, and aliquoted at hourly intervals for motility evaluation. Values are presented as the mean ± SEM (vertical bar) of six experiments



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  		FIG. 3. Effect of dilution on the motility of Indian white-backed vulture spermatozoa. Uncontaminated semen was diluted in TALP (black circle, 1:1; black square, 1:2; black triangle, 1:5; white square, 1:10), incubated at 37°C, and aliquoted at hourly intervals for motility evaluation. Values are presented as the mean ± SEM (vertical bar) of five experiments

Cryopreservation of Semen

Following cryopreservation of diluted vulture semen, postthaw motility was observed to be high (33%) in TALP containing 8% glycerol and 20% egg yolk compared to the other media (Table 2). Of the 33% motile spermatozoa, approximately 50–55% of these spermatozoa exhibited progressive and linear motility, like the spermatozoa from neat semen, but appeared to have reduced velocity. In fact, CASA analysis confirmed a decrease in the velocity parameters (VAP, VSL, and VCL) and a decrease in BCF, LIN, and STR in spermatozoa following cryopreservation and thawing compared to spermatozoa from normal semen (Table 3). However, the cryopreserved spermatozoa showed an increase in ALH.


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  		TABLE 2. Evaluation of glycerol and dimethyl sulphoxide as cryoprotec tants for cryopreservation of uncontaminated semen of the Indian white-backed vulture.a


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  		TABLE 3. Comparison of the motility parameters of the Indian white-backed vulture spermatozoa prior to cryopreservation and after cryopres ervation and thawing.a

Sperm Motility Kinetics by CASA

Sperm motility analysis was carried out in a slide chamber (depth, 130 µm) using CASA. The CASA system yielded consistent results using the conditions and parameter setup values described earlier [14]. In freshly diluted vulture semen, spermatozoa were extremely active and exhibited progressive motility along a linear trajectory. The movement of the tail was rapid, and the head of the spermatozoon was always pointed in the direction of the trajectory and exhibited lateral movements. The spermatozoa normally did not alter their direction of movement, except when they encountered an obstacle. Furthermore, even when the suspensions were incubated for prolonged periods of time (5 h), changes in the motility pattern were not observed except that they had become sluggish and the percentage of motile spermatozoa decreased. Table 4 shows the time-dependent changes in the motility parameters of vulture spermatozoa. The percentage of motile spermatozoa did not decrease during the first 2 h. The latter decreased significantly, however, and by the fifth hour, motility was only 20%.


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  		TABLE 4. Time dependent change in the motility parameter of the Indian white-backed vulture spermatozoa (n = 100) incubated in TALP at 37°C.*

Motility parameters, such as VAP, VSL, STR, ALH, and BCF, did not change between 0 h and the first hour but significantly decreased from the second to fifth hours. The straightness of the trajectory represented by STR did not show a significant difference in the first 3 h. Both VCL and LIN showed a significant decrease after the second hour of incubation.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
To our knowledge, this is the first known report of successful semen collection from the Indian white-backed vulture, an endangered species [2–4]. The present study demonstrates that the massage method of semen collection can be used to induce ejaculation in the Indian white-backed vulture. To date, three methods have been reported for semen collection in nondomestic birds: the cooperative method, in which birds such as falcons and raptors voluntarily ejaculate into special devices, such as an artificial vagina [9–12]; the electroejaculation method, as used in domestic ducks and geese [10]; and the massage method, as used in pigeons, pheasants, ducks, and so on [13]. In fact, the efficiency with which the vultures ejaculated (84.6% successful attempts) is comparable to the successful ejaculations obtained in other birds, such as American kestrels (74%) [25], sandhill cranes (86%) [10], and blue rock pigeons (90%) [14], using the same method. In the present study, 39.4% of the ejaculates were contaminated with urine or fecal material. Earlier studies also indicated urine as a common source of contamination of ejaculates from the golden eagle (36.8%), the imperial eagle (43.1%), the peregrine falcon (48.2%) [6], and American kestrels (50%) [25]. A side effect of the massage technique is that it stimulates urination [10]. Blanco et al. [6] reported that anatomical and behavioral differences in golden and imperial eagles were the reason for greater contamination of semen in these eagles compared to the Bonnelli eagle.

The ejaculate volume of the uncontaminated semen of the Indian white-backed vulture varied over a wide range (0.05–1.4 ml). Detailed analysis of the ejaculates from four different individuals, however, indicated that the volume variations were in the range of 0.05–0.6 ml except for one single ejaculate from vulture 577, which yielded 1.4 ml. This single ejaculate is, indeed, the cause of the skewed distribution observed with respect to ejaculate volume. Such variations in semen volume are not uncommon and have been observed in the Andean condor (0.05–0.45 ml) [26], turkey (0.2–0.5 ml) [27], and Houbara bustard (0.05– 0.25 ml) [28]. Furthermore, the average semen volume of 0.37 ml also is comparable with birds, such as pheasant (0.39 ml) [29], duck (0.3 ml) [30], and goose (0.01–0.6 ml) [31]. The average sperm concentration in the ejaculates (58.4 x 106 ml–1) was significantly lower than that of Turkey (8000 x 106 ml–1) [32], mallard duck (1320 x 106 ml–1) [33], pigeon (3200 x 106 ml–1) [14], and northern pintail (5700 x 106 ml–1) [34] but higher than that of the prairie falcon (20 x 106 ml–1) [35] and budgerigar (25 x 106 ml–1) [36]. The mean sperm motility (46.8%) is similar to those of the Bonelli eagle (32–39%) and golden eagle (30–33%) but lower than those of the imperial eagle (55– 62%) and peregrine falcon (55–59%) [6].

The pH of semen of Indian white-backed vulture was neutral (pH 7.1), thus closely resembling the semen of the Bonelli eagle (pH 7.18) and golden eagle (pH 7.25) [6]. Urine contamination made semen slightly acidic (pH 6.9), as reported earlier for the urine-contaminated semen of the Bonelli eagle (pH 6.86) and imperial eagle (pH 6.8) [6]. The presence of urine in the ejaculate severely reduced the proportion of motile sperm, as reported earlier [6, 37], but washing the contaminated ejaculates with a neutral diluent improved the percentage of motile spermatozoa. The white-backed vulture ejaculates contained a low percentage of abnormal spermatozoa (17.8%), similar to that reported in ostriches (17%) [38] and Houbara bustard (10–24%) [28]. However, a large proportion of the abnormal spermatozoa were macrocephalic (41%), and this type of abnormality is common in the golden eagle, peregrine falcon, and Houbara bustard [28]. Wishart et al., [28] suggested that the presence of macrocephalic sperm in Houbara bustard could be a genetically determined trait.

We found TALP to be the best semen extender compared to BPSE [15] and Lake diluent [16]. Blanco et al. [6] had used Lake extender to dilute and wash urine-contaminated semen from eagles and falcons, but in the present study, when Lake extender was used, the motility of spermatozoa declined drastically. Gee et al. [39] reported that BPSE, the commonly used semen extender for poultry, was better for the dilution of sandhill and whooping crane semen. However, in the present study, BPSE was observed to be less efficient as an extender compared to TALP.

Vulture semen, when diluted in the ratio of 1:1 with TALP, showed that more than 50% of the spermatozoa were motile by 2 h. This result is similar to the report of northern pintail [34], but best motility results for turkey [40], pheasants [41], and blue rock pigeon [14] were observed at 1:3, 1:3, and 1:100 dilution of semen, respectively. These results suggest a significant variation among the birds with reference to spermatozoal tolerance to dilution.

Cryobanking of spermatozoa in liquid nitrogen is useful for captive-breeding management and for long-term sperm banking of the founder stock [42]. Studies were attempted to cryopreserve white-backed vulture semen with the slow-fast freezing protocol used for pigeon [14]. The result showed that postthaw, 30–35% of the spermatozoa were motile. Room definitely exists for improvement to achieve a higher percentage of motile spermatozoa following cryopreservation and thawing. However, this may need extensive standardization using various diluents, cryoprotectants, and freezing regimes. Nevertheless, it may be worth mentioning that postthaw motility in the present study in the vultures was comparable to the results in Houbara bustard (24–42%) [43] but lower than that observed for blue rock pigeon (47%) [14].

Earlier, CASA was used to assess objectively sperm motility parameters, such as VAP, VCL, VSL, STR, LIN, BCF, and ALH, in many wild animals, such as lion, tiger [44], leopard [24], gazelle [45], and blue rock pigeon [14]. These earlier studies indicated an increase in VAP, VCL, VSL, STR, LIN, and BCF in rapidly swimming spermatozoa exhibiting linear trajectories. Therefore, ascertaining the above motility parameters could help to evaluate the motility status of spermatozoa with respect to its velocity and trajectory. In the present study, VAP, VSL, STR, and ALH did not change when incubated in TALP for up to 1 h but significantly decreased from the second hour onward, when they became sluggish and exhibited a curved trajectory. A similar observation was reported in the blue rock pigeon [14].

To our knowledge, this is the first report of semen collection from the Indian white-backed vultures, which is a critically endangered bird, using the massage method. Approximately 85% of the semen collection attempts yielded ejaculates. Semen characteristics, such as concentration of spermatozoa and percentage of motile spermatozoa, were low compared to those of other bird species. The present study provides basic information about semen collection procedure, semen characteristics, and short-term storage of semen of Indian white-backed vultures.


   ACKNOWLEDGMENTS
 
The authors would like to thank the Chief Wildlife Warden, Government of Andhra Pradesh, and the Curator of Nehru Zoological Park, Hyderabad, for permission to handle the animals in the zoo and V. Sivaram for technical help in the initial stages of the present study.


   FOOTNOTES
 
1 Supported by grants from the Central Zoo Authority of India and the Department of Biotechnology, Government of India. Back

2 Correspondence: S. Shivaji, Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India. FAX: 00 91 40 27160591; shivas{at}ccmb.res.in Back

Received: 3 May 2005.

First decision: 4 June 2005.

Accepted: 18 July 2005.


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 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
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