Zoo Biology 34: 335–344 (2015)
RESEARCH ARTICLE
Cryopreservation of Sambar Deer Semen in Thailand Thevin Vongpralub,1* Wittaya Chinchiyanond,2 Pornchai Hongkuntod,1 Pitcharat Sanchaisuriya,1 Sanan Liangpaiboon,2 Areeya Thongprayoon,2 and Noppadon Somphol1 1 2
Department of Animal Science, Faculty of Agriculture, Khon Kaen University, Khon Kaen, Thailand Wildlife Conservation Bureau, National Park, Wildlife and Plant Conservation Department, Bangkok, Thailand Little is known of the different freezing and thawing techniques for post-thaw survival of spermatozoa in Sambar deer. So, this study determined the effect of seminal plasma, egg yolk and glycerol extenders and their concentrations, plus cooling, freezing, and thawing protocols on the post-thaw quality of their semen. Semen samples were collected by electro-ejaculation from four Thai Sambar deer stags (Cervus unicolor equinus). As evaluated by post-thaw progressive motility and acrosome integrity removal of seminal plasma was beneficial; Tris-egg yolk was the most efficient extender; a 20% egg yolk concentration was better than the 0%, 10%, or 30%; and a 3% glycerol concentration was better than 5%, 7%, or 9%. Using the optimum dilution techniques, semen was loaded in 0.5 ml plastic straws. Cooling times from ambient temperature to 5°C in 3 hr resulted in higher post-thaw progressive motility and acrosome integrity than 1, 2, or 4 hr. Suspending the straws 4 cm above the surface for 15 min before plunging into liquid nitrogen was better than suspending at 2 or 6 cm. For thawing frozen semen, an intermediate thawing (50°C, 8 sec) protocol was more effective than the slower (37°C, 10 sec) or faster (70°C, 5 sec) thawing rates. Timed insemination following estrus synchronization of 10 hinds resulted in six confirmed pregnancies at 60 days. Five hinds delivered live fawns. This study provides an effective approach for semen cryopreservation and artificial insemination (AI), which should be valuable to scientists for genetics and reproductive management of Sambar deer © 2015 Wiley Periodicals, Inc. in developing countries. Zoo Biol. 34:335–344, 2015.
Keywords: tropical deer; frozen semen protocols; artificial insemination; field condition
INTRODUCTION Sambar deer (Cervus unicolor) are the largest species of oriental deer and are found throughout India, Southeast Asia, and Southern China. Of the 14 subspecies described by Whitehead [1993], Cervus unicolor equinus is the most abundant in Southeast Asia. Wild populations are rapidly declining in many Southeast Asian protected areas due to poaching [Leslie, 2011]. Sambar deer is the preferred deer meat in local and international markets [Steinmetz et al., 2006]. This species of deer attained slaughter weight earlier than red deer, suggesting that Sambar deer are worthly of domestication [Semiadi et al., 1995]. Therefore, captive Sambar deer farming has become commercially viable but good management requires a better understanding of the animal’s physiology. There is a general paucity of information on the reproductive physiology of mainland Southeast Asian Sambar deer, both in the wild and in captivity. In Thailand, female Sambar deer (Cervus unicolor equinus) estrus is not seasonal, normally 19–21 days estrous cycles and a gestation of 265.4 days are found [Chinchiyanoon, personal
© 2015 Wiley Periodicals, Inc.
communication (n ¼ 30)]. In comparison, the estrous cycle and gestation of female Formosan Sambar deer (Cervus unicolor swinhoei) were 18.2 and 258 days, respectively [Chan et al., 2009]. Sompon [2004] showed the semen of male Sambar deer in Thailand to have good quality during the rut and low semen quality when antlers were in velvet. As with domestic animals, cryopreservation of semen and artificial insemination (AI) has played a major role in conservation of genetic resources and propagation of wildlife Grant sponsor: National Park Wildlife and Plant Conservation Department. Correspondence to: Thevin Vongpralub, Department of Animal Science, Faculty of Agriculture, Khon Kaen University, Khon Kaen 40002, Thailand. E-mail: [email protected]
Received 08 May 2014; Revised 04 April 2015; Accepted 07 April 2015
DOI: 10.1002/zoo.21214 Published online 23 April 2015 in Wiley Online Library (wileyonlinelibrary.com).
336 Vongpralub et al. species. For captive populations, AI with frozen semen has the potential for overcoming inbreeding depression and maintaining genetic diversity. Reproductive techniques for deer have been adapted from small ruminants and cattle [Asher et al., 2000]. The tolerance of sperm to cryopreservation and the fertility of frozen-thawed sperm varies among species [Holt, 2000]. The survival of post-thaw spermatozoa is known to be affected by various factors such as diluent, cooling rate, cryoprotectant, equilibration time, freezing rate, and thawing temperature [Watson, 2000]. A few studies to date have examined sperm freezing and AI in the deer [Morrow et al., 2009]. Studies with frozen semen were reported for red deer, fallow deer, Pere David’s deer [Soler et al., 2003a,b], Iberian deer [Fernandez-Santos et al., 2006], and Iberian red deer [Martinez-Pastor et al., 2009]. Recently, Nally et al. [2011] compared the effect of glycerol concentration on the cryosurvival of Timor deer sperm. Until recently, there has been only one study to determine the effect of different diluents on post-thaw sperm quality in Formosan Sambar deer (Cervus unicolor swinhoie) and Formosan Sika deer (Cervus nippon taiouanua). That study found that different diluents required modification to preservation protocols for successful semen cryopreservation [Cheng et al., 2004]. Since specific protocols are required for optimal semen freezing for each breed or species, adequate knowledge is still lacking on the protocols for semen cryopreservation and AI in mainland Sambar deer. The objective of this study was to develop a practical cryopreservation protocol and AI technologies for the Sambar deer (Cervus unicolor equinus).
MATERIALS AND METHODS General Procedure
current intensity, from 0 to12 V, was divided in a 0–30 scale. Electrical stimuli began at a low voltage and was gradually increased until spermic ejaculates were obtained. Stimuli and rest periods were 2 sec each. All semen collections were conducted without anesthesia since use of anesthetics was prohibited by the Wildlife Breeding Research Center. Stags were manually restrained by experienced herdsmen under veterinarian supervision for an average 8.75 3.38 (4–15) min including an average of 3.86 2.66 min under operation time of electro-ejaculation. Quality of raw semen was assessed by volume, concentration, forward progressive motility (FPM), and normal sperm morphology [Evan and Maxwell, 1987]. The percentage of progressive motile sperm was estimated using a microscope. Ten microliters of semen was placed on a clean glass slide and covered with a coverslip. The semen sample was then examined on a warm (37°C) stage under a light microscope at 400 magnification at least 10 microscopic fields were examined. For sperm morphology assessment, one drop of semen sample was added to two drops of nigrosin eosin stain. Sperm morphology was determined by counting a total of 200 sperm under light microscope at 1,000 magnification. Sperm concentration was determined using a hemocytometer after dilution of raw semen (1:200) with 4% NaCl with a weak eosin solution. Only the semen with at least 60% FPM and 70% live normal sperm morphology was used in the experiments. The cryopreserved semen evaluation included FPM and acrosome integrity as described by Dott and Foster [1972]. For the assessment of intact acrosome of cryopreserved sperm, one drop of post-thaw semen was added to two drops of nigrosin-eosin stain. A drop of this mixture was smeared on microscope slide and the percentage of live (unstained) normal apical ridge sperm (Fig. 1) was immediately determined by counting a total of 200 sperm under light microscope at 1,000 magnification.
Animals Thai Sambar deer maintained at the Khao Kao Wildlife Breeding Research Center, Petchaboon, Thailand (16.66°N, 101.00°E, 700 m elevation) were used in this study. Four mature stags (2.5–5 years) and 10 multiparous hinds (5–7 years) were individually housed in outdoor pens (9 12 m) and exposed to a natural photoperiod and ambient temperature. Forage, mineral block, and water were provided ad libitum. All animals were fed daily a 16% crude protein concentrate mix of maize and soybean meal at 0.5% BW. Stags were hand-reared to facilitate for electro-ejaculation procedures. Semen collection and evaluation Semen (one ejaculate/stag/experiment) was collected between November and March, the period in which stags exhibit maximum breeding behavior. Electro-ejaculation was achieved using an Electrojac IV (Gemini Inc., MN) ejaculator with a rectal probe (2.5 cm diameter, 20.5 cm length) with three longitudinal electrodes. The electrical
Zoo Biology
Semen processing The semen extenders were maintained at 35°C in a water bath prior to collection of semen. After collection, the semen was centrifuged at 1,920g for 30 sec and the seminal plasma discarded, except for Experiment 1. The semen was then diluted with Tris-egg yolk extender [Salisbury et al., 1978], except for Experiment 2, to obtain a concentration of 20 106 cell/ml. The egg yolk concentration in the extender was 20% (v/v), except for Experiment 3. Diluted semen was cooled according to the method described previously [Evan and Maxwell, 1987] with minor modification. Briefly, extended semen in 15 ml centrifuge tube was transferred into 500 ml beaker containing 200 ml of 35°C water (working as a water jacket). Then, beaker was placed inside a styrofoam box containing 15°C water. Temperature was gradually lowered by adding water and ice into Styrofoam box to reach 5°C within 3 hr, except for Experiment 4. At 5°C, the diluted semen was re-diluted (1:1 v/v) with a 12% glycerolated diluent in order to achieve a final concentration
Cryopreservation of Sambar Deer Semen 337
Fig. 1. Acrosome of post-thaw Sambar deer spermatozoa. (A) Live sperm with intact acrosome. (B) Live sperm with damage acrosome. (C) Dead sperm.
of 6% glycerol, except for Experiment 5. Glycerolization was accomplished by a stepwise addition of four equal volumes of extender at 15 min intervals. Diluted semen was loaded into 0.5 ml plastic straws and held at 5°C for 2 hr for equilibration and then frozen in static liquid nitrogen vapor. Semen was frozen in styrofoam boxes (25.5 37.5 29.0 cm) containing at least 10 cm of liquid nitrogen. The 1 temperature was measured using TC 200 (Dickson, IL), a digital thermometer. The straws were placed in the vapor 4 cm above the liquid nitrogen, except for Experiment 6, for 15 min and then plunged into the liquid nitrogen. To assess post-thaw semen quality, straws were stored in liquid nitrogen for at least 14 days and then thawed in a 37°C water bath for 1 min, except for Experiment 7. Summary of conditions and treatments of the experiment 1–7 are shown in Table 1. Experimental Procedure Experiment 1: Effect of removal of seminal plasma on post-thaw semen quality Semen from four stags was divided into two aliquots and centrifuged. Seminal plasma was removed from the first portion and re-diluted with Tris-egg yolk to provide 20 106 progressively motile spermatozoa per milliliter. The second portion was directly diluted with Tris-egg yolk extender to obtain the same concentration of spermatozoa as the first one. Semen samples were processed for cryopreservation, thawed, and evaluated as described above. Experiment 2: Effect of different extenders on the postthaw quality of the semen A comparison of four egg yolk extenders (20%) included (1) Tris-egg yolk; (2) egg yolk citrate; (3) Illinois Variable Temperature (IVT) extender [Salisbury et al., 1978]; and (4) Beltsville F5 extender [Pursel and Johnson, 1975]. The ejaculate of each of the four stags was split into four portions. After dilution, the samples were centrifuged and the seminal plasma was discarded. Each of the four
sperm pellets was re-suspended in one of the four different extenders. Semen samples were processed, frozen, thawed, and evaluated as described above, except for the different egg yolk extenders. Experiment 3: Effect of egg yolk concentration of the extender on the post-thaw semen motility and acrosome integrity Semen from three stags was divided into four portions each, diluted, and centrifuged to remove the seminal plasma. (One of the four stags could not be used because of poor semen quality.) Subsequently, sperm pellets were resuspended with Tris-egg yolk extender containing 0%, 10%, 20%, and 30% (v/v) egg yolk. Semen samples were processed, frozen, thawed, and evaluated as described above, except for the different concentrations of extender. Experiment 4: Effect of different cooling rates on postthaw semen quality To evaluate the effect of cooling rate on post-thaw survival of spermatozoa, semen from three stags was processed, frozen, thawed, and evaluated as described above, except that the cooling rate, from 35°C to 5°C was accomplished over 1, 2, 3, or 4 hr. Semen samples of each treatment were cooled by adding water and small ice cubes manually into ice baths (Styrofoam container) as described above. The cooling rate of each treatment was controlled by the amount of ice and cold water. The cooling rates were approximately 0.41, 0.21, 0.14, and 0.10°C/min, respectively. Experiment 5: Effect of different glycerol concentrations on post-thaw semen quality To evaluate different glycerol concentrations the semen from three stags was processed, frozen, thawed, and evaluated as described above except glycerolization was achieved by adding the extender plus glycerol to achieve final dilutions of 3%, 5%, 7%, and 9% glycerol (v/v). Zoo Biology
338 Vongpralub et al. TABLE 1. Summary of conditions and treatments of the experiments 1–7 Conditions Objective to determine
Seminal plasma
1
Effect of seminal plasma
Yes
2
Effect of extender
Exp.
3
Effect of egg yolk
No Best of Exp. 1
Best of Exp. 1
Yolk (%)
Cooling rates
Tris- egg yolk
20
3
6
4
37, 1
Tris-egg yolk Egg yolk citrate IVT Beltsville F5 Best of Exp. 2
20
3
6
4
37, 1
3
6
4
37,1
1
6
4
37,1
3
4
37,1
2
37,1
4 6 Best of Exp. 6
37, 10 sec.
Best of Exp. 6
50, 8 sec. 70, 5 sec. Best of Exp. 7
Extender
concentration 4
5
6
7
8
Effect of cooling rates
Effect of glycerol concentration
Effect of freezing rates Effect of thawing temperature Frozen semen fertility test AI
Best of Exp. 1
Best of Exp. 1
Best of Exp. 1 Best of Exp. 1 Best of Exp. 1
Best of Exp. 2
Best of Exp. 2
Best of Exp. 2 Best of Exp. 2 Best of Exp. 2
Thawing temp. and time (°C, min)
20 20 20 0 10 20 30 Best of Exp. 3
Best of Exp. 3
Best of Exp. 3 Best of Exp. 3 Best of Exp. 3
2 3 4 Best of Exp. 4
Best of Exp. 4 Best of Exp. 4 Best of Exp. 4
Experiment 6: Effect of different freezing rates on the post-thaw semen quality To evaluate different freezing rates, semen from three stags was processed, frozen, thawed, and evaluated as described above, semen from each stage were divided to three groups and maintained horizontally in the liquid nitrogen vapor at 2, 4, and 6 cm above the liquid nitrogen, where the temperature at these levels were 180, 150, and 100°C, respectively. After exposure of straws in liquid nitrogen vapor for 15 min, they were immersed into liquid nitrogen. Experiment 7: Effect of thawing temperature and time on post-thaw semen quality To determine the effects of thawing temperature and time, the semen from three stags was processed, and frozen as described above. After storage for at least 14 days, the straws were thawed at 37°C for 10 sec, 50°C for 8 sec, and 70°C for 5 sec. The semen was evaluated as described above. Zoo Biology
Glycerol Straw locate above liquid (%) nitrogen (cm)
5 7 9 Best of Exp. 5 Best of Exp. 5 Best of Exp. 5
Experiment 8: Comparison of single and double AI with frozen semen on pregnancy rate Estrus was synchronized in 10 hinds using the controlled progesterone releasing intravaginal device for goats (CIDR-G, Inter Ag, Hamilton, New Zealand) for 13 days. At the time of removal, an IM injection of 200 IU equine Chorionic Gonadotropin (eCG) (Folligon; Intervet International BV, Holland) and PGF2a (375 mg cloprostenol; 1.5 ml estroplan; Parnell laboratories Pty, Ltd., Australia) were administered. Fives hinds were inseminated once 48 hr after CIDR-G withdrawal and the remaining five hinds were inseminated twice 48 and 60 hr after CIDR-G withdrawal. Protocol for timed insemination was determined by preliminary estrus synchronization studies (n ¼ 8) in which estrus was detected by closely observing the behavior of a trained, mature stag as he interacted with hinds in 12-hr intervals. Onset of estrus was determined when the hinds
Cryopreservation of Sambar Deer Semen 339
allowed a 5-min one-to-one interaction with the stag. Estrus was observed 30 hr after CIDR-G withdrawal and lasted 24–60 hr. The highest quality semen from single ejaculation from one stag was used to inseminate ten hinds. Quality was determined by semen volume, percentage of motile sperm, percentage of live sperm, percentage of live normal sperm, and sperm concentration which was 1 ml, 85%, 95%, 91%, and 1,400 million cells/ml, respectively. The best methods, as determined from the results of above experiments, were used to freeze semen with 20 106 cells/straw. Progressively motile sperm of the thawed semen was always more than 65% motility. For each insemination approximately 13 106 progressively motile sperm were used. To achieve insemination, a bovine breeding gun was inserted into a foam type catheter for pig AI (pig champ). The modified catheter was then inserted through the vagina, close to the cervix. Adjustments were then made to the tip of the catheter, and its correct position in the cervix was confirmed by rectal examination, so that the bovine gun could be inserted through the first ring of the cervix to deposit the semen. One time of AI, only one straw was used for each female. No anesthesia was used for AI since use of anesthetic drugs was not approved by the breeding center. All hinds were manually restrained with a soft net and under veterinarian inspection for an average time of 5.4 1.1 (4–7) min. Statistical Analysis Four randomly selected straws of semen from each stag (replicates) were assigned to each treatment (Experiments 1–7). Response variables were analyzed by ANOVA procedures of SAS (Ver.6; Statistical Analysis System Institute, Cary, NC) using a randomized complete block design where blocks were stags. Means for freezing treatments were separated by Duncan’s New Multiple Range Test, with level of significance set at P < 0.05, when Pvalues of the F-test were significant (P < 0.05). Conception rates in Experiment 8 were compared using the x2 test. RESULTS Fresh semen quality from 28 ejaculates varied as semen was collected from the four stags seven times during the collection period. Five ejaculates from one stag were discarded due to low quality and 23 were used in Experiments 1 through 7 (three to four semen samples for each experiment). Mean semen volume, motile sperm, live sperm, live normal sperm, and sperm concentration (mean SD) were 1.55 0.97 ml, 70.91 9.84%, 84.86 6.24%, 77.82 7.22%, and 477.05 255.32 million cells/ml, respectively, for all ejaculates. Experiment 1: Effects of removal of seminal plasma on post-thaw semen quality Seminal plasma had a detrimental effect on frozen Thai Sambar deer spermatozoa. Removal of the seminal plasma
increased the post-thawed progressive sperm motility at both 0 and 5 hr of incubation (increase from 39.5% to 47.9% at 0 hr and from 15.5% to 26.3% at 5 hr, P < 0.01). Also, the percentage of intact acrosomes increased from 35.5% to 47.9% at 0 hr (P < 0.01; see Table 2). Experiment 2: Effect of different extenders on the postthaw quality of the semen For Thai Sambar deer semen, Tris-egg yolk extender resulted in the highest post-thaw sperm progessive motility rates compared to egg yolk citrate, Illinois Variable Temperature (IVT), and Beltsville F5 extenders. (46.7%, 41.0%, 42.1%, and 35.2%, respectively, at 0 hr and 30.0%, 25.0%, 25.0%, and 21.3%, respectively, at 2 hr post-thaw (P < 0.05). Also, the use of Tris-egg yolk extender resulted in the greatest acrosome integrity (38.1% compared to 35.4%, 34.7% and 30.0%, respectively, at 0 hr post-thaw (P < 0.05). Egg yolk citrate and IVT extenders were not different (P > 0.05; see Table 3). Experiment 3: Effects of egg yolk concentration of the extender on the post-thaw semen quality and acrosome integrity For Thai Sambar deer semen, Tris-egg yolk diluent containing 20% (v/v) egg yolk yielded the best (P < 0.05) post-thaw sperm progressive motility. The 0%, 10%, 20%, and 30% concentrations of egg yolk resulted in 33.6%, 39.6%, 49.3%, and 35.5%, respectively, sperm progressive motility. Intact acrosomes were 33.2%, 38.4%, 43.7%, and 32.0%, respectively. The 0% and 30% concentrations of egg yolk were not different (P > 0.05; see Table 4). Experiment 4: Effects of different cooling rates on postthaw semen quality For Thai Sambar deer semen, 3 hr was the most beneficial cooling time (P < 0.05) from 35°C to 5°C prior to freezing. The 1, 2, 3, and 4 hr cooling times resulted in 19.58%, 21.17%, 28.25%, and 21.67%, respectively, sperm progressive motility. Intact acrosomes were 24.17%, 26.33%, 35.00%, and 27.75%, respectively. The 1, 2, and
TABLE 2. Effect of removal of seminal plasma on the postthaw quality of Sambar deer frozen semen (mean SD) Seminal plasma Parameters
present
Progressive motility (%) 0 hr 39.5a 3.90 5 hr 15.5a 4.59 Intact acrosome (%) 0 hr 35.5a 4.90
absent
SEM
47.9b 3.78 26.3b 4.69
0.96 1.16
47.9b 5.10
1.25
ab
Within rows, means without a common superscript differ (P < 0.05).
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340 Vongpralub et al. TABLE 3. Effects of different extenders on the post-thaw quality of Sambar deer frozen semen (mean SD) Extenders Parameters Progressive motility (%) 0 hr 2 hr Intact acrosome (%) 0 hr
Tris
Citrate
BF5
IVT
SEM
46.7a 7.79 30.0a 1.69
41.0b 1.71 25.0b 1.59
42.1b 1.77 25.0b 1.57
35.2c 1.94 21.3c 1.55
0.45 0.40
38.1a 1.11
35.4b 1.25
34.7b 1.20
30.0c 1.09
0.29
Tris, Tris-egg yolk extender; Citrate, sodium citrate egg yolk extender; BF5, Beltsville F5 extender; IVT, Illinois Variable Temperature extender. abcWithin rows, means without a common superscript differ (P < 0.05).
4 hr cooling times were not different for intact acrosome (P > 0.05; see Table 5). Experiment 5: Effect of different glycerol concentrations on post-thaw semen quality For Thai Sambar deer semen, 3% was the best concentration (P < 0.05) of glycerol for freezing semen. The 3%, 5%, 7%, and 9% concentrations of glycerol resulted in 37.8%, 28.9%, 25.1%, and 22.5%, respectively, sperm progressive motility. Intact acrosomes were 40.0%, 34.3%, 30.7%, and 24.7%, respectively (see Table 6). Experiment 6: Effect of different freezing rates on the post-thaw semen quality For Thai Sambar deer semen, 4 cm was the best height (P < 0.05) to suspend semen straws in the liquid nitrogen vapor before plunging into the liquid nitrogen. Estimate of cooling rates were 12, 10, and 5°C/min for 2, 4, and 6 cm heights, respectively. Final temperature at 2, 4, and 6 cm above nitrogen were 180, 150, and 100°C, respectively, and resulted in 34.04%, 42.96%, and 36.92%, respectively, sperm progressive motility. Intact acrosomes were 25.33%, 32.92%, and 27.92%, respectively (see Table 7). Experiment 7: Effect of thawing temperature and time on post-thaw semen quality For Sambar deer semen, an intermediate thawing rate (50°C for 8 sec) was better than the slow (37°C for 10 sec) or fast (70°C for 5 sec) thawing rates (P < 0.05). An intermediate thawing rate had a higher percentage of sperm progressive motility (52.08%) compared to slow (40.96%)
and fast (38.54%), respectively. In addition, intermediate thawing resulted in higher intact acrosomes (52.90%) compared to slow (40.00%) and fast (37.82%), respectively (see Table 8). Experiment 8: Comparison of single and double AI with frozen semen on pregnancy rate All 10 Sambar hinds (100%) exhibited estrus at 28.8 11.2 hr following removal of the CIDR-G and injections with 200 IU eCG and PGF2a. The duration of estrus was 28.0 6.1 hr. The hinds were manually restrained and duration of restraint including insemination for each hind was less than 10 min. Artificial insemination was performed in 2.8 1.0 min. Pregnancy rates and fawns born were not different when comparing single versus double timed AI (48 hr vs. 48 þ 12 hr, respectively) following estrus synchronization (P > 0.05). After 60 days, non-return to estrus was 60% for both treatments. The fawning rate for single AI was 40% versus 60% for double AI (P > 0.05; see Table 9).
DISCUSSION The present study was undertaken to develop a suitable protocol for semen cryopreservation of Sambar deer which could pave the way for their ex situ conservation as well as breeding and propagation. Eight experiments were carried out to provide an effective approach for semen cryopreservation and artificial insemination of Sambar deer. Based on these results, it could be inferred that seminal plasma should be removed before semen processing. Tris-egg yolk extender containing 20% egg yolk and 3% glycerol effectively maintained sperm quality after thawing. Best
TABLE 4. Effect of egg yolk concentration on post-thaw quality of Sambar deer frozen semen (mean SD) Level of Tris-egg yolk extender (%V/V) Parameter Progressive motility (%) Intact acrosome (%) abc
0
10
20
30
SEM
33.6c 3.00 33.2c 3.541
39.6b 3.25 38.4b 3.48
49.3a 2.97 43.7c 3.20
35.5c 3.27 32.0c 3.47
0.9 1.0
Within rows, means without a common superscript differ (P
RESEARCH ARTICLE
Cryopreservation of Sambar Deer Semen in Thailand Thevin Vongpralub,1* Wittaya Chinchiyanond,2 Pornchai Hongkuntod,1 Pitcharat Sanchaisuriya,1 Sanan Liangpaiboon,2 Areeya Thongprayoon,2 and Noppadon Somphol1 1 2
Department of Animal Science, Faculty of Agriculture, Khon Kaen University, Khon Kaen, Thailand Wildlife Conservation Bureau, National Park, Wildlife and Plant Conservation Department, Bangkok, Thailand Little is known of the different freezing and thawing techniques for post-thaw survival of spermatozoa in Sambar deer. So, this study determined the effect of seminal plasma, egg yolk and glycerol extenders and their concentrations, plus cooling, freezing, and thawing protocols on the post-thaw quality of their semen. Semen samples were collected by electro-ejaculation from four Thai Sambar deer stags (Cervus unicolor equinus). As evaluated by post-thaw progressive motility and acrosome integrity removal of seminal plasma was beneficial; Tris-egg yolk was the most efficient extender; a 20% egg yolk concentration was better than the 0%, 10%, or 30%; and a 3% glycerol concentration was better than 5%, 7%, or 9%. Using the optimum dilution techniques, semen was loaded in 0.5 ml plastic straws. Cooling times from ambient temperature to 5°C in 3 hr resulted in higher post-thaw progressive motility and acrosome integrity than 1, 2, or 4 hr. Suspending the straws 4 cm above the surface for 15 min before plunging into liquid nitrogen was better than suspending at 2 or 6 cm. For thawing frozen semen, an intermediate thawing (50°C, 8 sec) protocol was more effective than the slower (37°C, 10 sec) or faster (70°C, 5 sec) thawing rates. Timed insemination following estrus synchronization of 10 hinds resulted in six confirmed pregnancies at 60 days. Five hinds delivered live fawns. This study provides an effective approach for semen cryopreservation and artificial insemination (AI), which should be valuable to scientists for genetics and reproductive management of Sambar deer © 2015 Wiley Periodicals, Inc. in developing countries. Zoo Biol. 34:335–344, 2015.
Keywords: tropical deer; frozen semen protocols; artificial insemination; field condition
INTRODUCTION Sambar deer (Cervus unicolor) are the largest species of oriental deer and are found throughout India, Southeast Asia, and Southern China. Of the 14 subspecies described by Whitehead [1993], Cervus unicolor equinus is the most abundant in Southeast Asia. Wild populations are rapidly declining in many Southeast Asian protected areas due to poaching [Leslie, 2011]. Sambar deer is the preferred deer meat in local and international markets [Steinmetz et al., 2006]. This species of deer attained slaughter weight earlier than red deer, suggesting that Sambar deer are worthly of domestication [Semiadi et al., 1995]. Therefore, captive Sambar deer farming has become commercially viable but good management requires a better understanding of the animal’s physiology. There is a general paucity of information on the reproductive physiology of mainland Southeast Asian Sambar deer, both in the wild and in captivity. In Thailand, female Sambar deer (Cervus unicolor equinus) estrus is not seasonal, normally 19–21 days estrous cycles and a gestation of 265.4 days are found [Chinchiyanoon, personal
© 2015 Wiley Periodicals, Inc.
communication (n ¼ 30)]. In comparison, the estrous cycle and gestation of female Formosan Sambar deer (Cervus unicolor swinhoei) were 18.2 and 258 days, respectively [Chan et al., 2009]. Sompon [2004] showed the semen of male Sambar deer in Thailand to have good quality during the rut and low semen quality when antlers were in velvet. As with domestic animals, cryopreservation of semen and artificial insemination (AI) has played a major role in conservation of genetic resources and propagation of wildlife Grant sponsor: National Park Wildlife and Plant Conservation Department. Correspondence to: Thevin Vongpralub, Department of Animal Science, Faculty of Agriculture, Khon Kaen University, Khon Kaen 40002, Thailand. E-mail: [email protected]
Received 08 May 2014; Revised 04 April 2015; Accepted 07 April 2015
DOI: 10.1002/zoo.21214 Published online 23 April 2015 in Wiley Online Library (wileyonlinelibrary.com).
336 Vongpralub et al. species. For captive populations, AI with frozen semen has the potential for overcoming inbreeding depression and maintaining genetic diversity. Reproductive techniques for deer have been adapted from small ruminants and cattle [Asher et al., 2000]. The tolerance of sperm to cryopreservation and the fertility of frozen-thawed sperm varies among species [Holt, 2000]. The survival of post-thaw spermatozoa is known to be affected by various factors such as diluent, cooling rate, cryoprotectant, equilibration time, freezing rate, and thawing temperature [Watson, 2000]. A few studies to date have examined sperm freezing and AI in the deer [Morrow et al., 2009]. Studies with frozen semen were reported for red deer, fallow deer, Pere David’s deer [Soler et al., 2003a,b], Iberian deer [Fernandez-Santos et al., 2006], and Iberian red deer [Martinez-Pastor et al., 2009]. Recently, Nally et al. [2011] compared the effect of glycerol concentration on the cryosurvival of Timor deer sperm. Until recently, there has been only one study to determine the effect of different diluents on post-thaw sperm quality in Formosan Sambar deer (Cervus unicolor swinhoie) and Formosan Sika deer (Cervus nippon taiouanua). That study found that different diluents required modification to preservation protocols for successful semen cryopreservation [Cheng et al., 2004]. Since specific protocols are required for optimal semen freezing for each breed or species, adequate knowledge is still lacking on the protocols for semen cryopreservation and AI in mainland Sambar deer. The objective of this study was to develop a practical cryopreservation protocol and AI technologies for the Sambar deer (Cervus unicolor equinus).
MATERIALS AND METHODS General Procedure
current intensity, from 0 to12 V, was divided in a 0–30 scale. Electrical stimuli began at a low voltage and was gradually increased until spermic ejaculates were obtained. Stimuli and rest periods were 2 sec each. All semen collections were conducted without anesthesia since use of anesthetics was prohibited by the Wildlife Breeding Research Center. Stags were manually restrained by experienced herdsmen under veterinarian supervision for an average 8.75 3.38 (4–15) min including an average of 3.86 2.66 min under operation time of electro-ejaculation. Quality of raw semen was assessed by volume, concentration, forward progressive motility (FPM), and normal sperm morphology [Evan and Maxwell, 1987]. The percentage of progressive motile sperm was estimated using a microscope. Ten microliters of semen was placed on a clean glass slide and covered with a coverslip. The semen sample was then examined on a warm (37°C) stage under a light microscope at 400 magnification at least 10 microscopic fields were examined. For sperm morphology assessment, one drop of semen sample was added to two drops of nigrosin eosin stain. Sperm morphology was determined by counting a total of 200 sperm under light microscope at 1,000 magnification. Sperm concentration was determined using a hemocytometer after dilution of raw semen (1:200) with 4% NaCl with a weak eosin solution. Only the semen with at least 60% FPM and 70% live normal sperm morphology was used in the experiments. The cryopreserved semen evaluation included FPM and acrosome integrity as described by Dott and Foster [1972]. For the assessment of intact acrosome of cryopreserved sperm, one drop of post-thaw semen was added to two drops of nigrosin-eosin stain. A drop of this mixture was smeared on microscope slide and the percentage of live (unstained) normal apical ridge sperm (Fig. 1) was immediately determined by counting a total of 200 sperm under light microscope at 1,000 magnification.
Animals Thai Sambar deer maintained at the Khao Kao Wildlife Breeding Research Center, Petchaboon, Thailand (16.66°N, 101.00°E, 700 m elevation) were used in this study. Four mature stags (2.5–5 years) and 10 multiparous hinds (5–7 years) were individually housed in outdoor pens (9 12 m) and exposed to a natural photoperiod and ambient temperature. Forage, mineral block, and water were provided ad libitum. All animals were fed daily a 16% crude protein concentrate mix of maize and soybean meal at 0.5% BW. Stags were hand-reared to facilitate for electro-ejaculation procedures. Semen collection and evaluation Semen (one ejaculate/stag/experiment) was collected between November and March, the period in which stags exhibit maximum breeding behavior. Electro-ejaculation was achieved using an Electrojac IV (Gemini Inc., MN) ejaculator with a rectal probe (2.5 cm diameter, 20.5 cm length) with three longitudinal electrodes. The electrical
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Semen processing The semen extenders were maintained at 35°C in a water bath prior to collection of semen. After collection, the semen was centrifuged at 1,920g for 30 sec and the seminal plasma discarded, except for Experiment 1. The semen was then diluted with Tris-egg yolk extender [Salisbury et al., 1978], except for Experiment 2, to obtain a concentration of 20 106 cell/ml. The egg yolk concentration in the extender was 20% (v/v), except for Experiment 3. Diluted semen was cooled according to the method described previously [Evan and Maxwell, 1987] with minor modification. Briefly, extended semen in 15 ml centrifuge tube was transferred into 500 ml beaker containing 200 ml of 35°C water (working as a water jacket). Then, beaker was placed inside a styrofoam box containing 15°C water. Temperature was gradually lowered by adding water and ice into Styrofoam box to reach 5°C within 3 hr, except for Experiment 4. At 5°C, the diluted semen was re-diluted (1:1 v/v) with a 12% glycerolated diluent in order to achieve a final concentration
Cryopreservation of Sambar Deer Semen 337
Fig. 1. Acrosome of post-thaw Sambar deer spermatozoa. (A) Live sperm with intact acrosome. (B) Live sperm with damage acrosome. (C) Dead sperm.
of 6% glycerol, except for Experiment 5. Glycerolization was accomplished by a stepwise addition of four equal volumes of extender at 15 min intervals. Diluted semen was loaded into 0.5 ml plastic straws and held at 5°C for 2 hr for equilibration and then frozen in static liquid nitrogen vapor. Semen was frozen in styrofoam boxes (25.5 37.5 29.0 cm) containing at least 10 cm of liquid nitrogen. The 1 temperature was measured using TC 200 (Dickson, IL), a digital thermometer. The straws were placed in the vapor 4 cm above the liquid nitrogen, except for Experiment 6, for 15 min and then plunged into the liquid nitrogen. To assess post-thaw semen quality, straws were stored in liquid nitrogen for at least 14 days and then thawed in a 37°C water bath for 1 min, except for Experiment 7. Summary of conditions and treatments of the experiment 1–7 are shown in Table 1. Experimental Procedure Experiment 1: Effect of removal of seminal plasma on post-thaw semen quality Semen from four stags was divided into two aliquots and centrifuged. Seminal plasma was removed from the first portion and re-diluted with Tris-egg yolk to provide 20 106 progressively motile spermatozoa per milliliter. The second portion was directly diluted with Tris-egg yolk extender to obtain the same concentration of spermatozoa as the first one. Semen samples were processed for cryopreservation, thawed, and evaluated as described above. Experiment 2: Effect of different extenders on the postthaw quality of the semen A comparison of four egg yolk extenders (20%) included (1) Tris-egg yolk; (2) egg yolk citrate; (3) Illinois Variable Temperature (IVT) extender [Salisbury et al., 1978]; and (4) Beltsville F5 extender [Pursel and Johnson, 1975]. The ejaculate of each of the four stags was split into four portions. After dilution, the samples were centrifuged and the seminal plasma was discarded. Each of the four
sperm pellets was re-suspended in one of the four different extenders. Semen samples were processed, frozen, thawed, and evaluated as described above, except for the different egg yolk extenders. Experiment 3: Effect of egg yolk concentration of the extender on the post-thaw semen motility and acrosome integrity Semen from three stags was divided into four portions each, diluted, and centrifuged to remove the seminal plasma. (One of the four stags could not be used because of poor semen quality.) Subsequently, sperm pellets were resuspended with Tris-egg yolk extender containing 0%, 10%, 20%, and 30% (v/v) egg yolk. Semen samples were processed, frozen, thawed, and evaluated as described above, except for the different concentrations of extender. Experiment 4: Effect of different cooling rates on postthaw semen quality To evaluate the effect of cooling rate on post-thaw survival of spermatozoa, semen from three stags was processed, frozen, thawed, and evaluated as described above, except that the cooling rate, from 35°C to 5°C was accomplished over 1, 2, 3, or 4 hr. Semen samples of each treatment were cooled by adding water and small ice cubes manually into ice baths (Styrofoam container) as described above. The cooling rate of each treatment was controlled by the amount of ice and cold water. The cooling rates were approximately 0.41, 0.21, 0.14, and 0.10°C/min, respectively. Experiment 5: Effect of different glycerol concentrations on post-thaw semen quality To evaluate different glycerol concentrations the semen from three stags was processed, frozen, thawed, and evaluated as described above except glycerolization was achieved by adding the extender plus glycerol to achieve final dilutions of 3%, 5%, 7%, and 9% glycerol (v/v). Zoo Biology
338 Vongpralub et al. TABLE 1. Summary of conditions and treatments of the experiments 1–7 Conditions Objective to determine
Seminal plasma
1
Effect of seminal plasma
Yes
2
Effect of extender
Exp.
3
Effect of egg yolk
No Best of Exp. 1
Best of Exp. 1
Yolk (%)
Cooling rates
Tris- egg yolk
20
3
6
4
37, 1
Tris-egg yolk Egg yolk citrate IVT Beltsville F5 Best of Exp. 2
20
3
6
4
37, 1
3
6
4
37,1
1
6
4
37,1
3
4
37,1
2
37,1
4 6 Best of Exp. 6
37, 10 sec.
Best of Exp. 6
50, 8 sec. 70, 5 sec. Best of Exp. 7
Extender
concentration 4
5
6
7
8
Effect of cooling rates
Effect of glycerol concentration
Effect of freezing rates Effect of thawing temperature Frozen semen fertility test AI
Best of Exp. 1
Best of Exp. 1
Best of Exp. 1 Best of Exp. 1 Best of Exp. 1
Best of Exp. 2
Best of Exp. 2
Best of Exp. 2 Best of Exp. 2 Best of Exp. 2
Thawing temp. and time (°C, min)
20 20 20 0 10 20 30 Best of Exp. 3
Best of Exp. 3
Best of Exp. 3 Best of Exp. 3 Best of Exp. 3
2 3 4 Best of Exp. 4
Best of Exp. 4 Best of Exp. 4 Best of Exp. 4
Experiment 6: Effect of different freezing rates on the post-thaw semen quality To evaluate different freezing rates, semen from three stags was processed, frozen, thawed, and evaluated as described above, semen from each stage were divided to three groups and maintained horizontally in the liquid nitrogen vapor at 2, 4, and 6 cm above the liquid nitrogen, where the temperature at these levels were 180, 150, and 100°C, respectively. After exposure of straws in liquid nitrogen vapor for 15 min, they were immersed into liquid nitrogen. Experiment 7: Effect of thawing temperature and time on post-thaw semen quality To determine the effects of thawing temperature and time, the semen from three stags was processed, and frozen as described above. After storage for at least 14 days, the straws were thawed at 37°C for 10 sec, 50°C for 8 sec, and 70°C for 5 sec. The semen was evaluated as described above. Zoo Biology
Glycerol Straw locate above liquid (%) nitrogen (cm)
5 7 9 Best of Exp. 5 Best of Exp. 5 Best of Exp. 5
Experiment 8: Comparison of single and double AI with frozen semen on pregnancy rate Estrus was synchronized in 10 hinds using the controlled progesterone releasing intravaginal device for goats (CIDR-G, Inter Ag, Hamilton, New Zealand) for 13 days. At the time of removal, an IM injection of 200 IU equine Chorionic Gonadotropin (eCG) (Folligon; Intervet International BV, Holland) and PGF2a (375 mg cloprostenol; 1.5 ml estroplan; Parnell laboratories Pty, Ltd., Australia) were administered. Fives hinds were inseminated once 48 hr after CIDR-G withdrawal and the remaining five hinds were inseminated twice 48 and 60 hr after CIDR-G withdrawal. Protocol for timed insemination was determined by preliminary estrus synchronization studies (n ¼ 8) in which estrus was detected by closely observing the behavior of a trained, mature stag as he interacted with hinds in 12-hr intervals. Onset of estrus was determined when the hinds
Cryopreservation of Sambar Deer Semen 339
allowed a 5-min one-to-one interaction with the stag. Estrus was observed 30 hr after CIDR-G withdrawal and lasted 24–60 hr. The highest quality semen from single ejaculation from one stag was used to inseminate ten hinds. Quality was determined by semen volume, percentage of motile sperm, percentage of live sperm, percentage of live normal sperm, and sperm concentration which was 1 ml, 85%, 95%, 91%, and 1,400 million cells/ml, respectively. The best methods, as determined from the results of above experiments, were used to freeze semen with 20 106 cells/straw. Progressively motile sperm of the thawed semen was always more than 65% motility. For each insemination approximately 13 106 progressively motile sperm were used. To achieve insemination, a bovine breeding gun was inserted into a foam type catheter for pig AI (pig champ). The modified catheter was then inserted through the vagina, close to the cervix. Adjustments were then made to the tip of the catheter, and its correct position in the cervix was confirmed by rectal examination, so that the bovine gun could be inserted through the first ring of the cervix to deposit the semen. One time of AI, only one straw was used for each female. No anesthesia was used for AI since use of anesthetic drugs was not approved by the breeding center. All hinds were manually restrained with a soft net and under veterinarian inspection for an average time of 5.4 1.1 (4–7) min. Statistical Analysis Four randomly selected straws of semen from each stag (replicates) were assigned to each treatment (Experiments 1–7). Response variables were analyzed by ANOVA procedures of SAS (Ver.6; Statistical Analysis System Institute, Cary, NC) using a randomized complete block design where blocks were stags. Means for freezing treatments were separated by Duncan’s New Multiple Range Test, with level of significance set at P < 0.05, when Pvalues of the F-test were significant (P < 0.05). Conception rates in Experiment 8 were compared using the x2 test. RESULTS Fresh semen quality from 28 ejaculates varied as semen was collected from the four stags seven times during the collection period. Five ejaculates from one stag were discarded due to low quality and 23 were used in Experiments 1 through 7 (three to four semen samples for each experiment). Mean semen volume, motile sperm, live sperm, live normal sperm, and sperm concentration (mean SD) were 1.55 0.97 ml, 70.91 9.84%, 84.86 6.24%, 77.82 7.22%, and 477.05 255.32 million cells/ml, respectively, for all ejaculates. Experiment 1: Effects of removal of seminal plasma on post-thaw semen quality Seminal plasma had a detrimental effect on frozen Thai Sambar deer spermatozoa. Removal of the seminal plasma
increased the post-thawed progressive sperm motility at both 0 and 5 hr of incubation (increase from 39.5% to 47.9% at 0 hr and from 15.5% to 26.3% at 5 hr, P < 0.01). Also, the percentage of intact acrosomes increased from 35.5% to 47.9% at 0 hr (P < 0.01; see Table 2). Experiment 2: Effect of different extenders on the postthaw quality of the semen For Thai Sambar deer semen, Tris-egg yolk extender resulted in the highest post-thaw sperm progessive motility rates compared to egg yolk citrate, Illinois Variable Temperature (IVT), and Beltsville F5 extenders. (46.7%, 41.0%, 42.1%, and 35.2%, respectively, at 0 hr and 30.0%, 25.0%, 25.0%, and 21.3%, respectively, at 2 hr post-thaw (P < 0.05). Also, the use of Tris-egg yolk extender resulted in the greatest acrosome integrity (38.1% compared to 35.4%, 34.7% and 30.0%, respectively, at 0 hr post-thaw (P < 0.05). Egg yolk citrate and IVT extenders were not different (P > 0.05; see Table 3). Experiment 3: Effects of egg yolk concentration of the extender on the post-thaw semen quality and acrosome integrity For Thai Sambar deer semen, Tris-egg yolk diluent containing 20% (v/v) egg yolk yielded the best (P < 0.05) post-thaw sperm progressive motility. The 0%, 10%, 20%, and 30% concentrations of egg yolk resulted in 33.6%, 39.6%, 49.3%, and 35.5%, respectively, sperm progressive motility. Intact acrosomes were 33.2%, 38.4%, 43.7%, and 32.0%, respectively. The 0% and 30% concentrations of egg yolk were not different (P > 0.05; see Table 4). Experiment 4: Effects of different cooling rates on postthaw semen quality For Thai Sambar deer semen, 3 hr was the most beneficial cooling time (P < 0.05) from 35°C to 5°C prior to freezing. The 1, 2, 3, and 4 hr cooling times resulted in 19.58%, 21.17%, 28.25%, and 21.67%, respectively, sperm progressive motility. Intact acrosomes were 24.17%, 26.33%, 35.00%, and 27.75%, respectively. The 1, 2, and
TABLE 2. Effect of removal of seminal plasma on the postthaw quality of Sambar deer frozen semen (mean SD) Seminal plasma Parameters
present
Progressive motility (%) 0 hr 39.5a 3.90 5 hr 15.5a 4.59 Intact acrosome (%) 0 hr 35.5a 4.90
absent
SEM
47.9b 3.78 26.3b 4.69
0.96 1.16
47.9b 5.10
1.25
ab
Within rows, means without a common superscript differ (P < 0.05).
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340 Vongpralub et al. TABLE 3. Effects of different extenders on the post-thaw quality of Sambar deer frozen semen (mean SD) Extenders Parameters Progressive motility (%) 0 hr 2 hr Intact acrosome (%) 0 hr
Tris
Citrate
BF5
IVT
SEM
46.7a 7.79 30.0a 1.69
41.0b 1.71 25.0b 1.59
42.1b 1.77 25.0b 1.57
35.2c 1.94 21.3c 1.55
0.45 0.40
38.1a 1.11
35.4b 1.25
34.7b 1.20
30.0c 1.09
0.29
Tris, Tris-egg yolk extender; Citrate, sodium citrate egg yolk extender; BF5, Beltsville F5 extender; IVT, Illinois Variable Temperature extender. abcWithin rows, means without a common superscript differ (P < 0.05).
4 hr cooling times were not different for intact acrosome (P > 0.05; see Table 5). Experiment 5: Effect of different glycerol concentrations on post-thaw semen quality For Thai Sambar deer semen, 3% was the best concentration (P < 0.05) of glycerol for freezing semen. The 3%, 5%, 7%, and 9% concentrations of glycerol resulted in 37.8%, 28.9%, 25.1%, and 22.5%, respectively, sperm progressive motility. Intact acrosomes were 40.0%, 34.3%, 30.7%, and 24.7%, respectively (see Table 6). Experiment 6: Effect of different freezing rates on the post-thaw semen quality For Thai Sambar deer semen, 4 cm was the best height (P < 0.05) to suspend semen straws in the liquid nitrogen vapor before plunging into the liquid nitrogen. Estimate of cooling rates were 12, 10, and 5°C/min for 2, 4, and 6 cm heights, respectively. Final temperature at 2, 4, and 6 cm above nitrogen were 180, 150, and 100°C, respectively, and resulted in 34.04%, 42.96%, and 36.92%, respectively, sperm progressive motility. Intact acrosomes were 25.33%, 32.92%, and 27.92%, respectively (see Table 7). Experiment 7: Effect of thawing temperature and time on post-thaw semen quality For Sambar deer semen, an intermediate thawing rate (50°C for 8 sec) was better than the slow (37°C for 10 sec) or fast (70°C for 5 sec) thawing rates (P < 0.05). An intermediate thawing rate had a higher percentage of sperm progressive motility (52.08%) compared to slow (40.96%)
and fast (38.54%), respectively. In addition, intermediate thawing resulted in higher intact acrosomes (52.90%) compared to slow (40.00%) and fast (37.82%), respectively (see Table 8). Experiment 8: Comparison of single and double AI with frozen semen on pregnancy rate All 10 Sambar hinds (100%) exhibited estrus at 28.8 11.2 hr following removal of the CIDR-G and injections with 200 IU eCG and PGF2a. The duration of estrus was 28.0 6.1 hr. The hinds were manually restrained and duration of restraint including insemination for each hind was less than 10 min. Artificial insemination was performed in 2.8 1.0 min. Pregnancy rates and fawns born were not different when comparing single versus double timed AI (48 hr vs. 48 þ 12 hr, respectively) following estrus synchronization (P > 0.05). After 60 days, non-return to estrus was 60% for both treatments. The fawning rate for single AI was 40% versus 60% for double AI (P > 0.05; see Table 9).
DISCUSSION The present study was undertaken to develop a suitable protocol for semen cryopreservation of Sambar deer which could pave the way for their ex situ conservation as well as breeding and propagation. Eight experiments were carried out to provide an effective approach for semen cryopreservation and artificial insemination of Sambar deer. Based on these results, it could be inferred that seminal plasma should be removed before semen processing. Tris-egg yolk extender containing 20% egg yolk and 3% glycerol effectively maintained sperm quality after thawing. Best
TABLE 4. Effect of egg yolk concentration on post-thaw quality of Sambar deer frozen semen (mean SD) Level of Tris-egg yolk extender (%V/V) Parameter Progressive motility (%) Intact acrosome (%) abc
0
10
20
30
SEM
33.6c 3.00 33.2c 3.541
39.6b 3.25 38.4b 3.48
49.3a 2.97 43.7c 3.20
35.5c 3.27 32.0c 3.47
0.9 1.0
Within rows, means without a common superscript differ (P
