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Seasonal variations in semen characteristics, semen cryopreservation, estrus synchronization, and successful artificial insemination in the spotted deer (Axis axis)
Theriogenology 67 (2007) 1371–1378 www.theriojournal.com
Seasonal variations in semen characteristics, semen cryopreservation, estrus synchronization, and successful artificial insemination in the spotted deer (Axis axis) Govindhaswamy Umapathy, Sadanand D. Sontakke, Anuradha Reddy, S. Shivaji * Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India Received 26 October 2006; accepted 16 January 2007
Abstract Ten adult male spotted deer were monitored over a 2-year interval to determine seasonal variations in testicular size, semen characteristics and serum testosterone concentrations, and to determine if there was an association between season and type of antler. Mean (S.E.M.) testicular volume (118.8 4.6 cm3), serum testosterone concentration (1.2 0.1 ng/mL), semen volume (4.1 0.6 mL), sperm concentration (338.3 24.9 106 mL1), percentage of morphologically normal sperm (79.1 2.8%), and percentage of motile sperm (66.5 1.5%) were higher (P < 0.05) in hard antler deer (peaked from March to May) than in deer with velvet antlers or in deer in which the antler has been shed. Thus, March–May was considered the physiologic breeding season for these deer; at this time, all stags had hard antlers. Furthermore, a Tris-citrate-based semen extender containing 4% glycerol and 20% egg-yolk was adequate for cryopreservation of semen. Estrus was induced with an implant containing norgestomet, timed transcervical AI was done with fresh semen, and 3 of 10 females were pregnant at 60 days, with fawns born 120 (premature), 240 and 243 days after AI. These results were considered a model for the use of assisted reproductive techniques to conserve other critically endangered deer species of India. # 2007 Published by Elsevier Inc. Keywords: Spotted deer; Antler cycle; Semen; Estrus synchronization; Artificial insemination
1. Introduction Based on a recent IUCN report [1], 23% of the world’s mammals are under serious threat of extinction due to habitat loss and fragmentation, as well as poaching. In India, many wild animals, especially ungulates (e.g. Manipur-brow antler deer, Swamp deer and Musk deer) are highly endangered and their numbers in the wild have declined to a few hundred animals. Thus, there is an urgent need to use both in situ
* Corresponding author. Tel.: +91 40 27192504; fax: +91 40 27160591. E-mail address: [email protected] (S. Shivaji). 0093-691X/$ – see front matter # 2007 Published by Elsevier Inc. doi:10.1016/j.theriogenology.2007.01.019
and ex situ conservation strategies to prevent their extinction. The ex situ conservation measures include captive breeding and re-introduction of the animals into the wild. Modern scientific advancements and technologies have played a major role in the conservation of some critically endangered wild animals (e.g. Eld’s deer [2], Mouflon [3], and Spanish ibex [4]). As part of the conservation efforts of Indian endangered animals, we have undertaken several research programs to develop and standardize various assisted reproductive techniques [5–9]. The present study was conducted to successfully perform semen collection, semen cryopreservation, estrus synchronization, and AI in spotted deer as a model for other endangered deer species of India.
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Research on the spotted deer has been limited to a few studies on semen collection [10], one on AI [11], and a few related to the seasonality of reproduction [12,13]. It is noteworthy that all of these studies were conducted outside India and have limited details regarding semen characteristics in relation to day length and serum testosterone concentrations, nor do they have much information regarding semen cryopreservation, induction of estrus, and AI. Therefore, the objective of the present study was to study seasonal variation in semen characteristics and antler growth and to successfully conduct assisted reproductive techniques, including collection and cryopreservation of semen, estrus synchronization, and AI in the commonly available spotted deer. In that regard, this species was used as a model for other critically endangered deer species in India, including Musk deer, Manipur-Brow antler deer, and Swamp deer. 2. Materials and methods 2.1. Study area and climate The study was conducted between 2002 and 2004 at the Nehru Zoological park, Hyderabad, India, located at 178220 N latitude and 788270 E longitude, at an altitude of 530 m, with a semi-arid climate and an annual rainfall of 500 mm. The monthly minimum and maximum air temperatures ranged from 12 to 27 8C and from 21 to 44 8C, respectively. The day length ranged from 11.05 to13.10 h (Indian Meteorological Department, Government of India, New Delhi, India). 2.2. Deer Adult male (n = 10; weight, 30–40 kg) and female (n = 10; weight, 25–30 kg) spotted deer were captured in the wild and maintained at the Nehru Zoological Park, Hyderabad. They were individually identified with ear tags and group-housed (separately for the two sexes) in large outdoor enclosures (15 m 17 m) with a shelter (2 m 3 m). The deer had free access to shade, water, mineral blocks containing salt and essential minerals, and were fed green fodder (2–4 kg/(animals day)) and a commercial feed concentrate containing all essential nutrients (0.5 kg/(animals day)). Prior to the study, the deer were allowed at least 5 months to acclimate to their enclosures. All 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 also approved by the Institutional Animal
Ethics Committee of the Centre for Cellular and Molecular Biology, Hyderabad, India. 2.3. Anesthesia, testis measurements, and semen collection Stags were anesthetized with a combination of ketamine hydrochloride (2.5 mg/kg body weight) and xylazine hydrochloride (0.5 mg/kg) injected i.m. (delivered by blowpipe). Once they were in lateral recumbency, blood was collected from the jugular vein, and the length and width of the testes were measured with vernier calipers. Testis volume was estimated with the formula for a prolate spheroid [14]. Semen was collected using electroejaculation [5,6]; an electroejaculator and rectal probe were used to deliver 20–30 stimuli (2–4 V in pulses that were 3–6 s in duration) and semen was collected into 50 mL pre-warmed tubes (Tarson, Calcutta, India). Following semen collection, anesthesia was reversed with yohimibine hydrochloride (0.2 mg/kg body weight; Antagozil; Troy Laboratories, Australia) given i.v. Semen characteristics, testis volume, serum testosterone concentration, and antler status (velvet, hard or those deer in which the antler has been shed) were monitored in three males, two or three times every month for 2 years; similar data were obtained once monthly from the remaining seven stags. 2.4. Semen evaluation Following semen collection, semen volume was determined with a micropipette (Gilson, Roissey Ch De Gaulle, Cedex, France), semen pH with indicator strips (Qualigens Fine Chemicals, Glaxo India Ltd., Mumbai), and sperm concentration was determined with a Makler chamber. The percentage of progressively motile sperm was estimated (increments of 10%) with phase-contrast microscopy (400). For sperm morphology studies, 2 mL of neat semen was fixed in 100 mL of 0.5% gluteraldehyde, smeared on a glass slide, and 200 sperm were assessed with phase-contrast microscopy (400). 2.5. Semen cryopreservation Semen samples with >70% motility were diluted 1:1 either in TALP (Tyrode medium supplemented with albumin, lactate and pyruvate [7]) or in a Tris-citrate medium containing 20% egg-yolk and 4% or 8% glycerol (final concentration, 200–250 106 sperm/ mL at 37 8C). The extended semen was loaded into 0.25 mL straws (IMV, L’Aigle, France) and frozen in a programmable cryogenic unit (Consarctic, Gottingen
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Germany), as follows: 24–4 8C at 1 8C/min and then from 4 to –80 8C at 6 8C/min, followed by plunging into liquid nitrogen. Two or 3 days later, the straws were removed from the liquid nitrogen and thawed in a 37 8C water bath for 1 min and evaluated for post-thaw sperm motility.
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androstane-3a,17b-diol, 0.2% 5b-androstane-3b,17bdiol, 0.2% 5-androstano-3b,17b-diol, and 0.02% estradiol. The sensitivity of the assay was 0.04 ng/ mL and the inter- and intra-assay CVs were 7.3–11% and 5–15.5%, respectively. 2.8. Histology of the testis
2.6. Estrus induction and AI An estrus synchronization treatment marketed for cattle (Crestar; Intervet, Boxmeer, The Netherlands [3]) was used to induce estrus in the adult female spotted deer. The treatment consisted of an implant containing 3 mg of norgestomet that was inserted intradermally in the ear, with a solution containing 3 mg norgestomet and 5 mg estradiol valerate injected i.m. at implant insertion. After 10 days, the implant was removed and 200 IU of eCG (Folligon, Intervet, Boxmeer, The Netherlands) was given i.m. For AI, a surgical plane of anesthesia was induced by i.m. injection (delivered with a blowpipe) of ketamine hydrochloride and xylazine hydrochloride (1.0 and 1.5 mg/kg body weight, respectively) between 48 and 57 h after implant removal. For insemination, a speculum with a fiberoptic light source (Caprine Supply, Brockville, Ont., Canada) was inserted into the vagina to visualize the os cervix. A cattle AI pipette (outside diameter, 3 mm) fitted with a syringe containing freshly collected semen (approximately 400–450 106 sperm in 2 mL of TALP) was inserted into the os cervix, manipulated gently through the cervical canal until no further forward movement was possible, and the semen slowly deposited. Immediately after insemination, anesthesia was reversed with 0.2 mg/kg yohimbine hydrochloride given i.v. Sixty days after AI, pregnancy diagnosis was conducted with transabdominal, B-mode ultrasonography (5 MHz linear-array transducer; Para Vet Ultrasonography, Pie Medical, The Netherlands).
Testes were collected opportunistically from four stags (two in the hard antler and two in velvet antler stage) that had died accidentally (various reasons) in the zoo. The testes and epididymides were fixed in 10% buffered formalin, embedded in paraffin, sectioned (5 mm), stained with haematoxylin–eosin, and examined microscopically. 2.9. Statistical analysis Data on testis volume, serum testosterone concentrations and semen parameters were grouped into four seasons, viz., November–January, February–April, May–July, and August–October and a repeated-measures ANOVA (general linear model) was used to determine the effects of season, year, and the year by season interaction. For comparison of values between velvet and hard antler, and between cryopreservation protocols, Mann–Whitney U-tests were used. Spearman rank correlation (rs) was used to determine correlations among various end points, including testis volume, day length, serum testosterone concentrations, semen volume, sperm concentration, percentage of motile and morphologically abnormal sperm. All statistical analyses were performed using the statistical software program SPSS@ 11.1 Windows (SPSS Inc., Chicago, IL, USA). Data are presented as mean S.E.M. and P 0.05 was considered significant. 3. Results
2.7. Radioimmunoassay
3.1. Semen collection
Blood collected from the stags was allowed to clot and serum was separated by centrifugation (3000 g for 10 min). The serum was stored at 20 8C until assayed for testosterone. Testosterone concentrations were measured, in duplicate, with a commercial radioimmunoassay kit (Coat-a-Count TKTT2; Diagnostic Products Corporation, Los Angeles, CA, USA), in accordance with the manufacturer’s instructions. The antiserum had the following cross-reactivities: 100% testosterone, 3.3% 5a-dihydrotestosterone, 0.8% 11-bhydroxytestosterone, 0.5% androstenedione, 0.4% 5-b-
Semen was readily collected by electroejaculation; overall, 192 of 194 attempts (98.9%) yielded ejaculates. Usually four or five stimuli were sufficient to initiate ejaculation, and in most cases, extrusion of the penis was observed. Although the first fraction of the ejaculate was creamy or milky white in color, viscous and had a high concentration of sperm (500–800 106 sperm/mL), subsequent fractions were watery, colourless, less viscous and less concentrated (100–300 106 sperm/ mL). Semen concentrations were reported based on the average concentration of the pooled ejaculate.
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3.2. Semen characteristics Semen characteristics of 144 ejaculates from stags with hard antlers (mating phase) and 48 ejaculates from stags with velvet antlers (non-mating phase) are summarized (Table 1). There were significant differences between the two seasons for all endpoints, with the exception of semen pH (overall, 8.0 0.1). 3.3. Seasonal variation in testis volume, serum testosterone concentrations, semen characteristics, and antler status To detect seasonal variations in testis volume, serum testosterone concentrations, and semen characteristics (i.e. semen volume, sperm concentration, percentage of motile sperm, percentage of sperm with normal versus abnormal morphology) in stags, data obtained from 10 stages over a period of 2 years were pooled with reference to month (irrespective of the year of collection) and plotted against mean day length of the month (Fig. 1). Testis volume varied over time (F = 6.8; P < 0.001); testis volume was maximal from April to June (Fig. 1a), decreased by nearly 40% by September, and reached a nadir in November. Mean testis volume (118.8 cm3) was greater during the hard antler period (6–8 months) than during the velvet antler phase (74.6 cm3, n = 18; MW U, P < 0.001). There was an effect of season on serum testosterone concentrations (F = 6.1, P < 0.001). Testosterone concentrations (range, 0.4–3.0 ng/mL; Fig. 1b) were lowest from September to November (when antlers were shed) and highest from March to May, when full-grown antlers were present (MW, P < 0.001). Semen volume, sperm concentration and percentage of motile sperm (Fig. 1c– e) were highest from May to July (F = 2.4, F = 11.0, and F = 9.3; all P < 0.05), concurrent with long days (Fig. 1h). There was an effect of season on the percentage of abnormal sperm (F = 8.0, P < 0.001; Fig. 1f); the percentage of abnormal sperm was high when semen volume, sperm concentration, and percentage of motile sperm were low. Semen pH was not significantly affected by season (Fig. 1g). There was
neither significant effect of year nor season by year interaction for any end point. Testis volume was correlated with day length and serum testosterone (rs = 0.71, P < 0.001; rs = 0.56, P < 0.05, respectively). Semen volume, sperm concentration and percentage of motile sperm were positively correlated with testis volume (rs = 0.76, P < 0.001; rs = 0.81, P < 0.001; rs = 0.66, P < 0.001, respectively). All end points were correlated with mean day length (serum testosterone, rs = 0.04, P < 0.05; testis volume, rs = 0.72, P < 0.001; semen volume, rs = 0.56, P < 0.05; sperm concentration, rs = 0.45, P < 0.001; sperm motility, rs = 0.42, P < 0.001). 3.4. Cryopreservation of sperm Tris-citrate containing 4% glycerol with 20% eggyolk was the best extender for cryopreservation with 45% of the sperm exhibiting post-thaw motility and 75% of these were progressively motile (Table 2). Increasing glycerol to 8% or using TALP (with 4% or 8% glyclerol) significantly decreased percentages of both motile and progressively motile sperm (P < 0.05). 3.5. Artificial insemination Of the 10 females inseminated with fresh semen, three were pregnant at 60 days; two animals delivered live fawns (240 and 243 days), whereas the third delivered a live pre-mature fawn (120 days). 3.6. Histology of the testis In histological sections of hard antler testis, there were active seminiferous tubules with spermatogenesis and spermiogenesis, including spermatogonia, spermatocytes, spermatids, and sperm. Epididymides of hard antler deer contained stored sperm (Figs. 2a and 3a). In contrast, the sections of velvet antler testis (n = 2) had only primary spermatogonia and round spermatids and the epididymides appeared empty (Figs. 2b and 3b).
Table 1 Mean (S.E.M.) testis volume, serum testosterone concentration, and semen characteristics of spotted deer with velvet vs. hard antler Antler type
Testis volume (cm3)
Serum testosterone (ng/mL)
Semen volume (mL)
Sperm concentrationa (106 mL1)
Motile sperm (%)
Morphologically abnormal sperm (%)
Velvet (n = 48) Hard (n = 144)
74.6 4.4 118.8 4.6
0.6 0.1 1.2 0.1
3.2 0.2 4.1 9.6
57.3 12.4 338.3 24.9
29.9 4.9 66.5 1.5
43.8 4.1 20.9 1.9
For each end point, there is a difference between deer with velvet vs. hard antler (P < 0.001). a Average for the entire ejaculate.
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Fig. 1. Box plots of monthly mean values from 10 spotted deer: (a) testis volume, (b), serum testosterone concentration, (c) semen volume, (d) sperm concentration, (e) percentage of motile sperm, (f) percentage of morphologically abnormal sperm, and (g) pH of semen. Mean day length is also shown (h).
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Table 2 Mean (S.E.M.) motile and progressively motile frozen-thawed spotted deer sperm following cryopreservation with two semen extenders and two concentrations of glycerol (all extenders contained 20% egg-yolk) Semen extender
Glycerol (%)
Motile sperm (%)
Progressively motilitya (%)
TALP
4 8
31 0.6 a 25 2.1 b
65 1.4 a 51 2.6 b
Tris-citrate
4 8
45 3.1 c 28 0.9 d
75 3.2 c 56 3.5 d
Within a column, values without a common letters (a–d) differ (P < 0.05). a Proportion of motile sperm that were progressively motile.
4. Discussion To our knowledge, this is the first report of a longterm study on seasonal variation in semen characteristics and successful AI in spotted deer. Although this species is endemic to India, its sub-species are found elsewhere [10,15]. In the present study, stags with hard antlers were in the physiologic breeding season, as indicated by high values for testis volume, serum testosterone concentrations, sperm concentration, and number of motile and morphologically normal sperm. That there was a clear seasonal antler pattern and
reproductive cycle in the male spotted deer confirmed previous reports [10] of an association between semen characteristics and the testicular cycle in the spotted deer from in the UK (a temperate country), and also in other deer species, including the white-tailed deer [16], fallow deer [14], Reeves’ muntjac [17], red deer [18], roe deer [12], and pampas deer [13]. In the present study, testis volume reached a nadir during the no antler and non-rutting phase, but was maximal during the rutting season, consistent with earlier seasonal changes in testis volume in fallow deer, red deer and the spotted deer [10,14,18].
Fig. 2. Histology of the testis of spotted deer. (a) Deer with hard antler; seminiferous tubules have a large lumen, elongated spermatids, and sperm. (b) Deer with velvet antler; lumen diameter is smaller and primary spermatogonia and round spermatids are present (scale bar = 100 mm).
Fig. 3. Cauda epididymides of spotted deer. (a) Deer with hard antler; sperm are present. (b) Deer with velvet antler; few sperm are present (scale bar = 100 mm).
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In seasonal mammals, spermatogenesis ceases when the testis volume decreased to <25% of peak size; this was associated with complete arrest of spermatogenesis [19]. However, Willard and Randel [15] reported that despite seasonal variations in antlers and in testis volume in axis stags in Texas, USA, testicular activity and epididymal sperm reserves were detected throughout the year. The present study also confirmed that spermatogonia and elongated spermatids are characteristics of deer with velvet antlers, as observed earlier in red deer and spotted deer [10,18]. In the present study, the antler cycle was very synchronous and closely associated with day length, in contrast to an earlier report by Loudon and Curlewis [10]. This apparent contradiction may be attributed to the study area (the United Kingdom), which has a temperate climate and is thus different from the tropics. Similar observations were made in Pampas deer, a tropical species; when transferred to a temperate region, they had non-synchronized antler cycling [13]. Bronson [20] suggested that in temperate regions, deer are seasonal breeders and their antler cycle is significantly influenced by photoperiod, whereas in tropical countries, reproductive activity may not be influenced by photoperiod but by the annual rainfall pattern [20,21]. In contrast to these observations, many tropical deer have similar antler cycles (Reeves deer [17]; Eld’s deer [2]; Pudu [22]). The present findings are supported by a previous observation on wild populations of spotted deer in Guindy National Park, Chennai (approximately 700 km south of the present study area) where the majority of the rutting activity was detected between April and July, coincident with 91% of adult males having hard antler [23]. Sperm cryopreservation of sperm is helpful for conservation and breeding of endangered animals in captivity. In the present study, a Tris-citrate extender containing 4% glycerol and 20% egg-yolk was an adequate medium. These results were consistent with the findings in other deer species, including the Iberian red deer [24], Formosan Sika deer and Formosan Sambar deer [25]. Glycerol is considered to be the most effective cryoprotectant for sperm of many mammalian species [26], with an optimal concentration ranging from 4% to 10% (v/v), depending on the species [5,27,28]. Synchronization of estrus prior to AI is a wellestablished procedure in domestic farm animals, but has been used very sparingly for wild animals such as fallow deer [29], sika deer [30], white-tailed deer [31], red deer [32], and gerenuk [33]. One of the major difficulties using this procedure induction of estrus; this
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needs to be optimized for each species. In the present study, we used a commercial, norgestomet-based synchronization system and timed-AI with fresh semen. That 3 of 10 deer fawned compared favorably with fawning rates in the spotted deer 43% [11], but appeared to be somewhat less successful than in the red deer (50% [27]) and fallow deer (43–68% [29]). Although the present study provided proof of concept, clearly further work is needed to improve the technique and to better establish pregnancy rates. In summary, it was clear that male spotted deer were seasonal breeders, with the breeding season effectively synchronized by day length. During the breeding season, stags had hard antlers and maximal testis volume, which was positively correlated with semen volume, sperm concentration, percentage of motile sperm, and serum testosterone concentrations. Furthermore, a Tris-citrate semen extender (containing 4% glycerol and 20% egg-yolk) was adequate for semen cryopreservation. Three of 10 females given a progestin-based synchronization treatment and timedAI subsequently delivered live fawns. These results are a model for the use of assisted reproductive techniques to conserve other critically endangered deer species of India. Acknowledgements This study was funded by the Department of Biotechnology, Ministry of Science and Technology, Government of India; Department of Biotechnology, Government of Andhra Pradesh and the Central Zoo Authority of India, Ministry of Environment and Forests, Government of India. We thank the Chief Wildlife Warden of Andhra Pradesh and the Curator of Nehru Zoological Park, Hyderabad for granting permission to conduct research and for their cooperation. We also thank Drs. S.D. Kholkute and Shakeel Ahmed for technical help in the initial stages of the study. References [1] IUCN. IUCN red list of threatened species. A global species assessment IUCN. Switzerland and Cambridge: Gland; 2006. [2] Monfort SL, Asher GW, Wildt DE, Wood TC, Schiewe MC, Williamson LR, et al. Successful intrauterine insemination of Eld’s deer (Cervus eldi thamin) with frozen-thawed spermatozoa. J Reprod Fertil 1993;99:459–65. [3] Ptak G, Clinton M, Barboni B, Muzzeddu M, Cappai P, Tischner M, et al. Preservation of the Wild European Mouflon: the first example of genetic management using a complete program of reproductive biotechnologies. Biol Reprod 2002;66:796–801.
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[19] Lincoln GA. In: Drew K, Fennessy PF, editors. Seasonal breeding in deer biology of deer production, vol. 22. Wellington, Bull: Royal Society of New Zealand; 1985. p. 165. [20] Bronson FH. In: Bronson FH, editor. Mammalian reproductive biology. Chicago: The University of Chicago Press; 1989 . p. 7–28. [21] Mishra HR, Wemmer C. The comparative breeding ecology of four cervids in Royal Chitwan National Park. In: Wemmer C, editor. Biology and management of the Cervidae. Washington: Smithsonian Institutional Press; 1987. [22] Reyes E, Bubenik GA, Schams D, Lobos A, Enriquez R. Seasonal changes of testicular parameters in southern pudu (Pudu puda) in relationship to circannual variation of its reproductive hormones. Acta Theriogenol 1997;42:25–35. [23] Raman RS. Antler cycles and breeding seasonality of the chital Axis axis in southern India. J Bombay Nat History Soc 1998;95:377–80. [24] Martinez-Pastor F, Anel L, Guerra C, Alvarez M, Soler AJ, Garde JJ, Chamorro C, de Paz P. Seminal plasma improves cryopreservation of Iberian red deer epididymal sperm. Theriogenology 2006;66:1847–56. [25] Cheng FP, Wu J, Chan JP, Wang J, Fung H, Colenbrander B, et al. The effect of different extenders on post-thaw sperm survival, acrosomal integrity and longevity in cryopreserved semen of Formosan Sika deer and Formosan Sambar deer. Theriogenology 2004;61:1605–16. [26] Holt WV. Fundamental aspects of sperm cryobiology: the importance of species and individual differences. Theriogenology 2000;53:47–58. [27] Asher GW, Berg DK, Evans G. Storage of semen and artificial insemination in deer. Anim Reprod Sci 2000;62:195–211. [28] Patil B, Jayaprakash D, Shivaji S. Cryopreservation of semen of tigers and lions: computerized analysis of the motility parameters of the spermatozoa. Curr Sci 1998;75:930–5. [29] Marrow CJ, Asher GW, Macmillan KL. Oestrous synchronization in farmed fallow deer (Dama dama): effects of season, treatment duration, and the male on the efficacy of the intravaginal CIDR device. Anim Reprod Sci 1995;37:159–74. [30] Willard ST, Hughes Jr DM, Bringans M, Sasser RG, White DR, Jaques JT, et al. Artificial insemination, hybridization and pregnancy detection in sika deer (Cervus nippon). Theriogenology 1996;46:779–89. [31] Willard ST, Neuendroff DA, Lewis AW, Randel RD. A comparison of transvaginal artificial insemination procedures for use in commercially farmed deer. Small Rumin Res 2002;44: 135–40. [32] Bowers SD, Brown CG, Strauch TA, Gandy BS, Neuendorff DA, Randel RD, et al. Artificial insemination following observational versus electronic methods of estrus detection in red deer hinds (Cervus elephus). Theriogenology 2004;62:652–63. [33] Penfold LM, Monfort S, Wolfe BA, Citino SB, Wildt DE. Reproductive physiology and artificial insemination studies in wild and captive gerenuk (Litocranius walleri walleri). Reprod Fertil Dev 2005;17:707–14.
Seasonal variations in semen characteristics, semen cryopreservation, estrus synchronization, and successful artificial insemination in the spotted deer (Axis axis) Govindhaswamy Umapathy, Sadanand D. Sontakke, Anuradha Reddy, S. Shivaji * Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India Received 26 October 2006; accepted 16 January 2007
Abstract Ten adult male spotted deer were monitored over a 2-year interval to determine seasonal variations in testicular size, semen characteristics and serum testosterone concentrations, and to determine if there was an association between season and type of antler. Mean (S.E.M.) testicular volume (118.8 4.6 cm3), serum testosterone concentration (1.2 0.1 ng/mL), semen volume (4.1 0.6 mL), sperm concentration (338.3 24.9 106 mL1), percentage of morphologically normal sperm (79.1 2.8%), and percentage of motile sperm (66.5 1.5%) were higher (P < 0.05) in hard antler deer (peaked from March to May) than in deer with velvet antlers or in deer in which the antler has been shed. Thus, March–May was considered the physiologic breeding season for these deer; at this time, all stags had hard antlers. Furthermore, a Tris-citrate-based semen extender containing 4% glycerol and 20% egg-yolk was adequate for cryopreservation of semen. Estrus was induced with an implant containing norgestomet, timed transcervical AI was done with fresh semen, and 3 of 10 females were pregnant at 60 days, with fawns born 120 (premature), 240 and 243 days after AI. These results were considered a model for the use of assisted reproductive techniques to conserve other critically endangered deer species of India. # 2007 Published by Elsevier Inc. Keywords: Spotted deer; Antler cycle; Semen; Estrus synchronization; Artificial insemination
1. Introduction Based on a recent IUCN report [1], 23% of the world’s mammals are under serious threat of extinction due to habitat loss and fragmentation, as well as poaching. In India, many wild animals, especially ungulates (e.g. Manipur-brow antler deer, Swamp deer and Musk deer) are highly endangered and their numbers in the wild have declined to a few hundred animals. Thus, there is an urgent need to use both in situ
* Corresponding author. Tel.: +91 40 27192504; fax: +91 40 27160591. E-mail address: [email protected] (S. Shivaji). 0093-691X/$ – see front matter # 2007 Published by Elsevier Inc. doi:10.1016/j.theriogenology.2007.01.019
and ex situ conservation strategies to prevent their extinction. The ex situ conservation measures include captive breeding and re-introduction of the animals into the wild. Modern scientific advancements and technologies have played a major role in the conservation of some critically endangered wild animals (e.g. Eld’s deer [2], Mouflon [3], and Spanish ibex [4]). As part of the conservation efforts of Indian endangered animals, we have undertaken several research programs to develop and standardize various assisted reproductive techniques [5–9]. The present study was conducted to successfully perform semen collection, semen cryopreservation, estrus synchronization, and AI in spotted deer as a model for other endangered deer species of India.
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Research on the spotted deer has been limited to a few studies on semen collection [10], one on AI [11], and a few related to the seasonality of reproduction [12,13]. It is noteworthy that all of these studies were conducted outside India and have limited details regarding semen characteristics in relation to day length and serum testosterone concentrations, nor do they have much information regarding semen cryopreservation, induction of estrus, and AI. Therefore, the objective of the present study was to study seasonal variation in semen characteristics and antler growth and to successfully conduct assisted reproductive techniques, including collection and cryopreservation of semen, estrus synchronization, and AI in the commonly available spotted deer. In that regard, this species was used as a model for other critically endangered deer species in India, including Musk deer, Manipur-Brow antler deer, and Swamp deer. 2. Materials and methods 2.1. Study area and climate The study was conducted between 2002 and 2004 at the Nehru Zoological park, Hyderabad, India, located at 178220 N latitude and 788270 E longitude, at an altitude of 530 m, with a semi-arid climate and an annual rainfall of 500 mm. The monthly minimum and maximum air temperatures ranged from 12 to 27 8C and from 21 to 44 8C, respectively. The day length ranged from 11.05 to13.10 h (Indian Meteorological Department, Government of India, New Delhi, India). 2.2. Deer Adult male (n = 10; weight, 30–40 kg) and female (n = 10; weight, 25–30 kg) spotted deer were captured in the wild and maintained at the Nehru Zoological Park, Hyderabad. They were individually identified with ear tags and group-housed (separately for the two sexes) in large outdoor enclosures (15 m 17 m) with a shelter (2 m 3 m). The deer had free access to shade, water, mineral blocks containing salt and essential minerals, and were fed green fodder (2–4 kg/(animals day)) and a commercial feed concentrate containing all essential nutrients (0.5 kg/(animals day)). Prior to the study, the deer were allowed at least 5 months to acclimate to their enclosures. All 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 also approved by the Institutional Animal
Ethics Committee of the Centre for Cellular and Molecular Biology, Hyderabad, India. 2.3. Anesthesia, testis measurements, and semen collection Stags were anesthetized with a combination of ketamine hydrochloride (2.5 mg/kg body weight) and xylazine hydrochloride (0.5 mg/kg) injected i.m. (delivered by blowpipe). Once they were in lateral recumbency, blood was collected from the jugular vein, and the length and width of the testes were measured with vernier calipers. Testis volume was estimated with the formula for a prolate spheroid [14]. Semen was collected using electroejaculation [5,6]; an electroejaculator and rectal probe were used to deliver 20–30 stimuli (2–4 V in pulses that were 3–6 s in duration) and semen was collected into 50 mL pre-warmed tubes (Tarson, Calcutta, India). Following semen collection, anesthesia was reversed with yohimibine hydrochloride (0.2 mg/kg body weight; Antagozil; Troy Laboratories, Australia) given i.v. Semen characteristics, testis volume, serum testosterone concentration, and antler status (velvet, hard or those deer in which the antler has been shed) were monitored in three males, two or three times every month for 2 years; similar data were obtained once monthly from the remaining seven stags. 2.4. Semen evaluation Following semen collection, semen volume was determined with a micropipette (Gilson, Roissey Ch De Gaulle, Cedex, France), semen pH with indicator strips (Qualigens Fine Chemicals, Glaxo India Ltd., Mumbai), and sperm concentration was determined with a Makler chamber. The percentage of progressively motile sperm was estimated (increments of 10%) with phase-contrast microscopy (400). For sperm morphology studies, 2 mL of neat semen was fixed in 100 mL of 0.5% gluteraldehyde, smeared on a glass slide, and 200 sperm were assessed with phase-contrast microscopy (400). 2.5. Semen cryopreservation Semen samples with >70% motility were diluted 1:1 either in TALP (Tyrode medium supplemented with albumin, lactate and pyruvate [7]) or in a Tris-citrate medium containing 20% egg-yolk and 4% or 8% glycerol (final concentration, 200–250 106 sperm/ mL at 37 8C). The extended semen was loaded into 0.25 mL straws (IMV, L’Aigle, France) and frozen in a programmable cryogenic unit (Consarctic, Gottingen
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Germany), as follows: 24–4 8C at 1 8C/min and then from 4 to –80 8C at 6 8C/min, followed by plunging into liquid nitrogen. Two or 3 days later, the straws were removed from the liquid nitrogen and thawed in a 37 8C water bath for 1 min and evaluated for post-thaw sperm motility.
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androstane-3a,17b-diol, 0.2% 5b-androstane-3b,17bdiol, 0.2% 5-androstano-3b,17b-diol, and 0.02% estradiol. The sensitivity of the assay was 0.04 ng/ mL and the inter- and intra-assay CVs were 7.3–11% and 5–15.5%, respectively. 2.8. Histology of the testis
2.6. Estrus induction and AI An estrus synchronization treatment marketed for cattle (Crestar; Intervet, Boxmeer, The Netherlands [3]) was used to induce estrus in the adult female spotted deer. The treatment consisted of an implant containing 3 mg of norgestomet that was inserted intradermally in the ear, with a solution containing 3 mg norgestomet and 5 mg estradiol valerate injected i.m. at implant insertion. After 10 days, the implant was removed and 200 IU of eCG (Folligon, Intervet, Boxmeer, The Netherlands) was given i.m. For AI, a surgical plane of anesthesia was induced by i.m. injection (delivered with a blowpipe) of ketamine hydrochloride and xylazine hydrochloride (1.0 and 1.5 mg/kg body weight, respectively) between 48 and 57 h after implant removal. For insemination, a speculum with a fiberoptic light source (Caprine Supply, Brockville, Ont., Canada) was inserted into the vagina to visualize the os cervix. A cattle AI pipette (outside diameter, 3 mm) fitted with a syringe containing freshly collected semen (approximately 400–450 106 sperm in 2 mL of TALP) was inserted into the os cervix, manipulated gently through the cervical canal until no further forward movement was possible, and the semen slowly deposited. Immediately after insemination, anesthesia was reversed with 0.2 mg/kg yohimbine hydrochloride given i.v. Sixty days after AI, pregnancy diagnosis was conducted with transabdominal, B-mode ultrasonography (5 MHz linear-array transducer; Para Vet Ultrasonography, Pie Medical, The Netherlands).
Testes were collected opportunistically from four stags (two in the hard antler and two in velvet antler stage) that had died accidentally (various reasons) in the zoo. The testes and epididymides were fixed in 10% buffered formalin, embedded in paraffin, sectioned (5 mm), stained with haematoxylin–eosin, and examined microscopically. 2.9. Statistical analysis Data on testis volume, serum testosterone concentrations and semen parameters were grouped into four seasons, viz., November–January, February–April, May–July, and August–October and a repeated-measures ANOVA (general linear model) was used to determine the effects of season, year, and the year by season interaction. For comparison of values between velvet and hard antler, and between cryopreservation protocols, Mann–Whitney U-tests were used. Spearman rank correlation (rs) was used to determine correlations among various end points, including testis volume, day length, serum testosterone concentrations, semen volume, sperm concentration, percentage of motile and morphologically abnormal sperm. All statistical analyses were performed using the statistical software program SPSS@ 11.1 Windows (SPSS Inc., Chicago, IL, USA). Data are presented as mean S.E.M. and P 0.05 was considered significant. 3. Results
2.7. Radioimmunoassay
3.1. Semen collection
Blood collected from the stags was allowed to clot and serum was separated by centrifugation (3000 g for 10 min). The serum was stored at 20 8C until assayed for testosterone. Testosterone concentrations were measured, in duplicate, with a commercial radioimmunoassay kit (Coat-a-Count TKTT2; Diagnostic Products Corporation, Los Angeles, CA, USA), in accordance with the manufacturer’s instructions. The antiserum had the following cross-reactivities: 100% testosterone, 3.3% 5a-dihydrotestosterone, 0.8% 11-bhydroxytestosterone, 0.5% androstenedione, 0.4% 5-b-
Semen was readily collected by electroejaculation; overall, 192 of 194 attempts (98.9%) yielded ejaculates. Usually four or five stimuli were sufficient to initiate ejaculation, and in most cases, extrusion of the penis was observed. Although the first fraction of the ejaculate was creamy or milky white in color, viscous and had a high concentration of sperm (500–800 106 sperm/mL), subsequent fractions were watery, colourless, less viscous and less concentrated (100–300 106 sperm/ mL). Semen concentrations were reported based on the average concentration of the pooled ejaculate.
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3.2. Semen characteristics Semen characteristics of 144 ejaculates from stags with hard antlers (mating phase) and 48 ejaculates from stags with velvet antlers (non-mating phase) are summarized (Table 1). There were significant differences between the two seasons for all endpoints, with the exception of semen pH (overall, 8.0 0.1). 3.3. Seasonal variation in testis volume, serum testosterone concentrations, semen characteristics, and antler status To detect seasonal variations in testis volume, serum testosterone concentrations, and semen characteristics (i.e. semen volume, sperm concentration, percentage of motile sperm, percentage of sperm with normal versus abnormal morphology) in stags, data obtained from 10 stages over a period of 2 years were pooled with reference to month (irrespective of the year of collection) and plotted against mean day length of the month (Fig. 1). Testis volume varied over time (F = 6.8; P < 0.001); testis volume was maximal from April to June (Fig. 1a), decreased by nearly 40% by September, and reached a nadir in November. Mean testis volume (118.8 cm3) was greater during the hard antler period (6–8 months) than during the velvet antler phase (74.6 cm3, n = 18; MW U, P < 0.001). There was an effect of season on serum testosterone concentrations (F = 6.1, P < 0.001). Testosterone concentrations (range, 0.4–3.0 ng/mL; Fig. 1b) were lowest from September to November (when antlers were shed) and highest from March to May, when full-grown antlers were present (MW, P < 0.001). Semen volume, sperm concentration and percentage of motile sperm (Fig. 1c– e) were highest from May to July (F = 2.4, F = 11.0, and F = 9.3; all P < 0.05), concurrent with long days (Fig. 1h). There was an effect of season on the percentage of abnormal sperm (F = 8.0, P < 0.001; Fig. 1f); the percentage of abnormal sperm was high when semen volume, sperm concentration, and percentage of motile sperm were low. Semen pH was not significantly affected by season (Fig. 1g). There was
neither significant effect of year nor season by year interaction for any end point. Testis volume was correlated with day length and serum testosterone (rs = 0.71, P < 0.001; rs = 0.56, P < 0.05, respectively). Semen volume, sperm concentration and percentage of motile sperm were positively correlated with testis volume (rs = 0.76, P < 0.001; rs = 0.81, P < 0.001; rs = 0.66, P < 0.001, respectively). All end points were correlated with mean day length (serum testosterone, rs = 0.04, P < 0.05; testis volume, rs = 0.72, P < 0.001; semen volume, rs = 0.56, P < 0.05; sperm concentration, rs = 0.45, P < 0.001; sperm motility, rs = 0.42, P < 0.001). 3.4. Cryopreservation of sperm Tris-citrate containing 4% glycerol with 20% eggyolk was the best extender for cryopreservation with 45% of the sperm exhibiting post-thaw motility and 75% of these were progressively motile (Table 2). Increasing glycerol to 8% or using TALP (with 4% or 8% glyclerol) significantly decreased percentages of both motile and progressively motile sperm (P < 0.05). 3.5. Artificial insemination Of the 10 females inseminated with fresh semen, three were pregnant at 60 days; two animals delivered live fawns (240 and 243 days), whereas the third delivered a live pre-mature fawn (120 days). 3.6. Histology of the testis In histological sections of hard antler testis, there were active seminiferous tubules with spermatogenesis and spermiogenesis, including spermatogonia, spermatocytes, spermatids, and sperm. Epididymides of hard antler deer contained stored sperm (Figs. 2a and 3a). In contrast, the sections of velvet antler testis (n = 2) had only primary spermatogonia and round spermatids and the epididymides appeared empty (Figs. 2b and 3b).
Table 1 Mean (S.E.M.) testis volume, serum testosterone concentration, and semen characteristics of spotted deer with velvet vs. hard antler Antler type
Testis volume (cm3)
Serum testosterone (ng/mL)
Semen volume (mL)
Sperm concentrationa (106 mL1)
Motile sperm (%)
Morphologically abnormal sperm (%)
Velvet (n = 48) Hard (n = 144)
74.6 4.4 118.8 4.6
0.6 0.1 1.2 0.1
3.2 0.2 4.1 9.6
57.3 12.4 338.3 24.9
29.9 4.9 66.5 1.5
43.8 4.1 20.9 1.9
For each end point, there is a difference between deer with velvet vs. hard antler (P < 0.001). a Average for the entire ejaculate.
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Fig. 1. Box plots of monthly mean values from 10 spotted deer: (a) testis volume, (b), serum testosterone concentration, (c) semen volume, (d) sperm concentration, (e) percentage of motile sperm, (f) percentage of morphologically abnormal sperm, and (g) pH of semen. Mean day length is also shown (h).
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Table 2 Mean (S.E.M.) motile and progressively motile frozen-thawed spotted deer sperm following cryopreservation with two semen extenders and two concentrations of glycerol (all extenders contained 20% egg-yolk) Semen extender
Glycerol (%)
Motile sperm (%)
Progressively motilitya (%)
TALP
4 8
31 0.6 a 25 2.1 b
65 1.4 a 51 2.6 b
Tris-citrate
4 8
45 3.1 c 28 0.9 d
75 3.2 c 56 3.5 d
Within a column, values without a common letters (a–d) differ (P < 0.05). a Proportion of motile sperm that were progressively motile.
4. Discussion To our knowledge, this is the first report of a longterm study on seasonal variation in semen characteristics and successful AI in spotted deer. Although this species is endemic to India, its sub-species are found elsewhere [10,15]. In the present study, stags with hard antlers were in the physiologic breeding season, as indicated by high values for testis volume, serum testosterone concentrations, sperm concentration, and number of motile and morphologically normal sperm. That there was a clear seasonal antler pattern and
reproductive cycle in the male spotted deer confirmed previous reports [10] of an association between semen characteristics and the testicular cycle in the spotted deer from in the UK (a temperate country), and also in other deer species, including the white-tailed deer [16], fallow deer [14], Reeves’ muntjac [17], red deer [18], roe deer [12], and pampas deer [13]. In the present study, testis volume reached a nadir during the no antler and non-rutting phase, but was maximal during the rutting season, consistent with earlier seasonal changes in testis volume in fallow deer, red deer and the spotted deer [10,14,18].
Fig. 2. Histology of the testis of spotted deer. (a) Deer with hard antler; seminiferous tubules have a large lumen, elongated spermatids, and sperm. (b) Deer with velvet antler; lumen diameter is smaller and primary spermatogonia and round spermatids are present (scale bar = 100 mm).
Fig. 3. Cauda epididymides of spotted deer. (a) Deer with hard antler; sperm are present. (b) Deer with velvet antler; few sperm are present (scale bar = 100 mm).
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In seasonal mammals, spermatogenesis ceases when the testis volume decreased to <25% of peak size; this was associated with complete arrest of spermatogenesis [19]. However, Willard and Randel [15] reported that despite seasonal variations in antlers and in testis volume in axis stags in Texas, USA, testicular activity and epididymal sperm reserves were detected throughout the year. The present study also confirmed that spermatogonia and elongated spermatids are characteristics of deer with velvet antlers, as observed earlier in red deer and spotted deer [10,18]. In the present study, the antler cycle was very synchronous and closely associated with day length, in contrast to an earlier report by Loudon and Curlewis [10]. This apparent contradiction may be attributed to the study area (the United Kingdom), which has a temperate climate and is thus different from the tropics. Similar observations were made in Pampas deer, a tropical species; when transferred to a temperate region, they had non-synchronized antler cycling [13]. Bronson [20] suggested that in temperate regions, deer are seasonal breeders and their antler cycle is significantly influenced by photoperiod, whereas in tropical countries, reproductive activity may not be influenced by photoperiod but by the annual rainfall pattern [20,21]. In contrast to these observations, many tropical deer have similar antler cycles (Reeves deer [17]; Eld’s deer [2]; Pudu [22]). The present findings are supported by a previous observation on wild populations of spotted deer in Guindy National Park, Chennai (approximately 700 km south of the present study area) where the majority of the rutting activity was detected between April and July, coincident with 91% of adult males having hard antler [23]. Sperm cryopreservation of sperm is helpful for conservation and breeding of endangered animals in captivity. In the present study, a Tris-citrate extender containing 4% glycerol and 20% egg-yolk was an adequate medium. These results were consistent with the findings in other deer species, including the Iberian red deer [24], Formosan Sika deer and Formosan Sambar deer [25]. Glycerol is considered to be the most effective cryoprotectant for sperm of many mammalian species [26], with an optimal concentration ranging from 4% to 10% (v/v), depending on the species [5,27,28]. Synchronization of estrus prior to AI is a wellestablished procedure in domestic farm animals, but has been used very sparingly for wild animals such as fallow deer [29], sika deer [30], white-tailed deer [31], red deer [32], and gerenuk [33]. One of the major difficulties using this procedure induction of estrus; this
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needs to be optimized for each species. In the present study, we used a commercial, norgestomet-based synchronization system and timed-AI with fresh semen. That 3 of 10 deer fawned compared favorably with fawning rates in the spotted deer 43% [11], but appeared to be somewhat less successful than in the red deer (50% [27]) and fallow deer (43–68% [29]). Although the present study provided proof of concept, clearly further work is needed to improve the technique and to better establish pregnancy rates. In summary, it was clear that male spotted deer were seasonal breeders, with the breeding season effectively synchronized by day length. During the breeding season, stags had hard antlers and maximal testis volume, which was positively correlated with semen volume, sperm concentration, percentage of motile sperm, and serum testosterone concentrations. Furthermore, a Tris-citrate semen extender (containing 4% glycerol and 20% egg-yolk) was adequate for semen cryopreservation. Three of 10 females given a progestin-based synchronization treatment and timedAI subsequently delivered live fawns. These results are a model for the use of assisted reproductive techniques to conserve other critically endangered deer species of India. Acknowledgements This study was funded by the Department of Biotechnology, Ministry of Science and Technology, Government of India; Department of Biotechnology, Government of Andhra Pradesh and the Central Zoo Authority of India, Ministry of Environment and Forests, Government of India. We thank the Chief Wildlife Warden of Andhra Pradesh and the Curator of Nehru Zoological Park, Hyderabad for granting permission to conduct research and for their cooperation. We also thank Drs. S.D. Kholkute and Shakeel Ahmed for technical help in the initial stages of the study. References [1] IUCN. IUCN red list of threatened species. A global species assessment IUCN. Switzerland and Cambridge: Gland; 2006. [2] Monfort SL, Asher GW, Wildt DE, Wood TC, Schiewe MC, Williamson LR, et al. Successful intrauterine insemination of Eld’s deer (Cervus eldi thamin) with frozen-thawed spermatozoa. J Reprod Fertil 1993;99:459–65. [3] Ptak G, Clinton M, Barboni B, Muzzeddu M, Cappai P, Tischner M, et al. Preservation of the Wild European Mouflon: the first example of genetic management using a complete program of reproductive biotechnologies. Biol Reprod 2002;66:796–801.
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