Reproductive parameters of donkey jacks undergoing puberty.
Rota Alessandraa*, Puddu Battistaa, Sabatini Chiaraa, Panzani Duccioa, Lainé Anne-Lyseb, Camillo Francescoa.
aDipartimento di Scienze Veterinarie, Università di Pisa, via livornese lato monte, 56122 San
Piero a Grado (PI), Italy.
bPRC, INRA, CNRS, IFCE, Université de Tours, 37380, Nouzilly, France
*Corresponding Author: Alessandra Rota E-mail: alessandra.rota@unipi.it
ABSTRACT
In male donkeys, puberty and the related events have been poorly characterized. The aim of this study was to evaluate the age at which male donkeys reach puberty, and characterize age associated changes in testicular size, testicular blood flow, serum testosterone concentration and semen quality. Every two months, starting at 6 months and finishing at 24 months of age, five male donkeys were subjected to B-mode ultrasound examination to assess testicular size and scrotum content and blood serum sampling for testosterone concentration. From the age of 8 months, pulsed Doppler was employed to evaluate blood flow in the testicular artery. Testosterone serum concentration was evaluated via RIA. From the age of 12 months, monthly semen collections were attempted and semen was evaluated for sperm number, motility and morphology. Onset of puberty was defined as the first ejaculate containing ≥50x106 spermatozoa with ≥10% total motility. Testes size was affected by age (P<0.0001) and after an initial plateau increased linearly from 10 months of age. Pulsatility and resistivity indexes were also affected by age (P<0.01), being significantly higher at 14 months than at 24 months. Testosterone serum concentration was affected by age (P<0.0001) and was significantly lower at 6-8 months (<0.2 ng/ml) compared to 22-24 months (≥0.8 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
ng/ml). Spermatozoa appeared in the ejaculate at a mean age of 18.9 months and puberty was attained between 19 and 22 months of age (mean:19.9 months), between January and March. In conclusion, late spring born Amiata donkey colts reached puberty at 20 months of age. Puberty was accompanied by changes in testicular size, testicular blood flow and serum testosterone concentration.
Keywords: Donkey; Puberty; Testosterone; Spermatozoa; Doppler
1. Introduction
In Europe the donkey has been the livestock species most affected by industrialization, depopulation of rural districts and mechanization of agriculture; this has led to a population decline of up to 80% in the twentieth century. Today, most of world donkey breeds either lack a population census, or are endangered or critical (Food and Agricultural Organization, 2014). Interest in donkey breeding, however, is now increasing both in an attempt to preserve biodiversity and for novel uses in the fields of milk production, social activities (onotherapy) and tourism (trekking). Semen preservation and artificial insemination may be used both to preserve precious genetic material and as an aid to breeders, in order to reduce inbreeding and improve selection of desired traits. For these reasons knowledge of donkey jacks’ reproductive physiology, including age at which they reach puberty and changes occurring during that time are of importance.
Puberty is the transition period following an increase in frequency and amplitude of GnRH secretion. It is completed when animals are able to release gametes, to show sexual behavior, and to successfully reproduce (Hafez, 1993; Senger, 2003). Spermatogenesis is dependent on the presence of testosterone and other hormones. In stallions, puberty was considered attained either when testosterone significantly increased (Brown-Douglas et al., 2004; Dhakal et al., 2012) or, as in bulls (Wolf et al., 1965), used the criteria 50x106 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49
spermatozoa with a motility ≥10% in the ejaculate (Naden et al., 1990). The first statistically significant rise in testosterone appeared in spring, at the age of 7-8 or 11-12 months in autumn or spring-born colts, respectively (Brown-Douglas et al., 2004; Dhakal et al., 2012). Semen collections, however, were not attempted in these studies. Naden et al. (1990) described two serum testosterone rises, a small one at 40-50 weeks and a dramatic one at 75-80 weeks, during the spring of the animal’s second year, and 50x106 spermatozoa with a motility ≥10% were present in the ejaculates at 83±2.9 weeks (range: 13-26 months) (Naden et al., 1990). This was in agreement with observations in Quarter Horses (12-23.6 months), although the mean age described in this breed was lower (55 weeks) (Cornwell, 1972). To our knowledge there are no studies on testosterone concentration and ejaculate characteristics during development and puberty of donkey foals, thus it is not known when they may first be employed as studs.
Testicular growth is also dependent on the endocrinological changes occurring during the pubertal period. In stallions, this growth has been described to occur from 42 weeks of age and to continue linearly thereafter, up to 96 weeks (Naden et al., 1990). In donkeys, the average volume of testicular parenchyma increased three folds from 1.5 to 5 years of age (Nipken and Wrobel, 1996), however nothing is knows about earlier changes. B-mode ultrasonography allows examination of the scrotal content, it can also be used to take precise measurements of the testes and evaluate testes structure and presence of abnormalities (Love 1992). The association of color and pulsed Doppler ultrasound enables evaluation of vascularization and blood flow. Physiological factors affecting testicular artery blood flow, including age have been evaluated in stallions (for a review see Ortega-Ferrusola et al., 2014). In humans it has been shown that resistivity index is lower in adults comparedto pre-pubertal children (Paltiel et al., 1994). Whether this is true for equids, and thus whether Doppler could be used to evaluate peri-pubertal events, is not known, as no such studies have been performed in pre and post-pubertal colts or donkey foals.
50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75
The aim of this study was to evaluate the age at which male donkeys reach puberty, and to characterize age associated changes in testicular size, testicular blood flow, serum testosterone concentration and semen quality.
2. Materials and methods
2.1. Animals
The study included five Amiata male donkeys, born between May and July 2012 and kept from birth and for the period of the study at the Department of Veterinary Sciences, Pisa University. During the period of the study the foals were maintained all together in a paddock, under natural weather and light conditions, and fed with hay ad libitum and horse food (Equifioc, Molitoria Val di Serchio, Lucca, Italy). The study was conducted between November 2012 and June 2014. The protocol was approved by the Ethical committee of the University of Pisa (prot. 14875, 2012).
2.2. Study outline
Donkeys were divided into two groups, one with birth date normalized at May 23rd, 2012 (n=2, range 12 days) and one with birth date normalized at June 24th, 2012 (n=3, range 22 days). Between 6 and 24 months of normalized age, each animal was subjected every two months (on the 22nd of each month, ±2.36 days) to blood sampling for hormonal analysis and ultrasound examination of the testes. From the age of 12 months, semen collections were attempted every 20-30 days until puberty was observed, and continued until the end of the study. Ejaculates were evaluated for presence, number and quality of sperm cells.
2.3. Ultrasound examinations
For ultrasound examination, animals were taken to an examination room, sedated with 40 mg/100 kg of xylazine, i.v. (Megaxilor, Bio98 Srl, Milano, Italy) and evaluated by B-mode 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100
and flow Doppler ultrasonography by Esaote MyLabTM30 Gold VET equipped with the 5-7.5 MHz linear probe Esaote LV513 VET (Esaote SpA, Firenze, Italy).
In B-mode, testicular width was measured, and testes, epididymides and spermatic cords were evaluated for presence of abnormalities or lesions. Measurements were taken from three images for each testis, and mean values were used for statistical analyses. For flow Doppler evaluation, each testicular artery was identified at its supratesticular, convoluted position and the lumen was detected with the aid of color flow mapping (CFM). Thereafter, the pulsed wave (PW) function was activated and flow diagrams were obtained and saved. Once transferred onto a PC, with the aid of the software MyLab Desk, spectral waves were manually identified and flux parameters and indexes were automatically calculated. Values for systolic peak velocity (SPV), end diastolic velocity (EDV), mean velocity (MV), pulsatility index (PI) and resistivity index (RI) were recorded and evaluated. Also in PW Doppler, when possible, three evaluations were performed, at an angle of insonation <60°. The set up for PW Doppler evaluation was kept constant throughout the study and was the following: Frequency 5MHz; PRF 2.8 KHz; Gain 64%; Wall filter 100 Hz; Sample gate 1 mm.
2.4. Hormonal analyses
At the time of the ultrasound examinations, blood samples were taken from the jugular vein, placed in serum sample tubes, and allowed to clot for 30 min. Serum was separated by centrifugation (3000 rpm for 10 min) and frozen at -18°C until evaluation. The concentrations of testosterone were determined using a direct radio-immunoassay method (Hochereau-De Reviers et al., 1990). For each sample, 100µl of plasma was assayed. To each tube, 350 µl buffer containing 7,5nCi labelled testosterone, antiserum at a dilution of 1/111 111 and 0.5 µg rabbit gamma-globulins was added. After incubation for 1h at 37°C, 50 µl of anti-rabbit gamma-globulin sheep serum was added. The samples were kept at 4°C overnight. The following day, 2 ml of polyethylene glycol at 80 g/l was added and the tubes were centrifuged 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126
(45 min, 4°C, 3200 rpm). After elimination of the supernatant, ethanol (100 µl/tube) and 2 ml of scintillation liquid were then added. The level of radioactivity was measured by a beta liquid scintillation counter. The antiserum cross-reacted with dihydrotestosterone (43%), androstenediol (4.5%), androstenedione (4.5%) androstanediol (3.5%) and less with other steroids (0.1%). The sensitivity of the assay was 0.0625 ng/ml. The intra-assay and inter-assay coefficients of variation were 5.6% and 5.9% at 0.5 ng/ml.
2.5. Semen collection and evaluation
Semen was collected in an open space, using a Missouri artificial vagina and the jack donkey mounting an estrous jenny. Immediately after collection, semen was filtered through sterile gauze. The volume after filtration was registered and two drops of semen were evaluated under a phase contrast microscope for presence of spermatozoa. When no sperm cells were observed, a smear stained with Diff-Quik® (Dade Behring, DE) was prepared to confirm their absence. When sperm cells were present, subjective motility was evaluated under a phase contrast microscope at 37°C on undiluted samples when concentration was low or after dilution in INRA96® at higher concentrations. Sperm concentration was evaluated using a Thoma counting chamber. Smears for sperm morphology (Spermac stain, Minitube, DE) and for presence of other cells (Diff-Quik®) were also prepared for each ejaculate.
Puberty was considered attained when at least 50x106 spermatozoa with ≥10% motility were present in the ejaculate (Wolf et al., 1965; Naden et al., 1990).
2.6. Statistical analyses
Data were evaluated for normality with the Anderson Darling test. Data on testicular size and flow Doppler were evaluated using the mean values obtained from the right and the left testes. Values on systolic peak velocity, normally distributed, were evaluated by one way ANOVA for repeated measures, including the effect of age and donkey. Dunnet’s multiple comparisons test was used for post hoc multiple comparisons. Data for testicular size and other values obtained from the Doppler analysis were evaluated by the non parametric 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152
Friedman’s test for repeated measures. Dunn’s multiple comparisons test was used for post hoc multiple comparisons. The same method was used for the evaluation of testosterone serum concentration. Age at semen collections was calculated from the birth date of the individual donkeys. Prism 6 for Windows (GraphPad software, Inc) was used for statistical analysis and P<0.05 was considered statistically significant.
3. Results
3.1 Ultrasound examinations
Testicular width was significantly affected by age (P<0.0001); after an initial plateau (up to 10 months), it increased progressively through the study. Statistically significant changes were observed between 6 to 10 and 22-24 months, and between 12 and 24 months of age (P<0.05; Fig. 1).
In three of the young donkeys’ testes no abnormalities were observed during the ultrasound examinations. In one animal a small anechoic structure (a testicular cyst or a tubular ectasia of the rete testis), localized at the cranial pole of the left testis, was constantly seen between 8 and 24 months, and during this time its diameter increased from 4.2 to 6.9 mm (Supplemental Fig. 1). In the fifth donkey, bilateral hydrocele was observed from month 14 onward (Supplemental Fig. 2). The data related to testicular artery blood flow of this last animal were not included in the data analyses as it showed a diastolic retrograde blood flow (Supplemental Fig. 3), not observed in the other animals of the same age, affecting PI and RI of both testes (Supplemental Fig. 4). Blood flow at six months was difficult to evaluate and only limited data were collected, thus results are described from 8 months of age only.
There was a significant effect of age (P=0.001), but not of donkey, on SPV (Table 1). Considering successive evaluations, a statistically significant increase of SPV was seen only 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177
between 12 and 14 months of age (May-June and July-August). Moreover, SPV at 24 months was significantly higher than SPV at 8, 10, 12, 16 and 18 months (P<0.05).
A significant effect of age (P<0.001) was also seen for EDV and MV. Considering successive evaluations, no statistically significant differences were seen for these parameters, nevertheless EDV was statistically higher at 24 months of age than at months 10 and 12, when it was always lower than 1 cm/sec (Table 1). A similar pattern was observed for MV, significantly higher at 24 months compared to months 8 and 10.
Pulsatility and resistivity indexes were also affected by age (P<0.01). PI was significantly higher at 14 months than at 24 months (Table 1). Similarly, RI was significantly higher at 12 and 14 months than at 24 months (Table 1). These data arise from the different spectral waves observed at 12-14 and at 24 months, with no diastolic flow in the younger animals (Fig. 2).
3.2. Hormonal analyses
Testosterone serum concentration was affected by age (P<0.0001), however the only statistically significant differences were observed between 6-8 and 22-24 months (from <0.2 ng/ml to ≥0.8 ng/ml, Fig. 3). There was a transient although not significant increase in serum testosterone at the age of 14 months (July-August, median 0.71, range 0.52-1.30).
3.3. Semen collection and evaluation
The first ejaculate collection was possible at a mean age of 15.2 months, however the first spermatozoa in the ejaculate appeared at 18.9 months and puberty, considered as the time when at least 50 million spermatozoa with ≥10% motility were present in the ejaculate, was reached at 19.9 months (Table 2). Mean sperm number at this time was 147.3±131.8 x106, while at 24 months it was 2.652±3.308 x106. Also motility was quite low at the first appearance of spermatozoa (<20% in 4/5 donkeys), while at 24 months it was 44±28% (range: 20-70%). Sperm morphology was first evaluated at 21-22 months (in February) when 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202
mean proportion of normal spermatozoa was 12%±6%, which increased at 24 months to 35.4±24.1%.
4. Discussion
This is the first comprehensive study on male donkey puberty, which was attained between 19 and 22 months of age, with a mean age of 19.9 months. The donkeys included in this study were born between May 29th and July 4th 2012, and all became pubertal between January and March 2014, thus after their second winter solstice and when daylight length was increasing. Four donkeys showed normal mounting behavior and ejaculated in the artificial vagina after their first winter solstice (July-August 2013), from the age of 13-15 months. At this time there was also a distinct, although not statistically significant, increase in testosterone, which probably stimulated the mounting behavior. The samples collected, however, were still azoospermic. In horses, Naden et al. (1990) described how 7/15 foals born between July 10th and September 3rd reached puberty (i.e. ≥50x106 spermatozoa with ≥10% motility) during the first trimester of the year after their second winter solstice (January-March). In the same study, two foals were pubertal earlier (September and November, at age of 56 and 68 weeks only) and six later (April-June) (Naden et al., 1990). In another study, puberty, defined as the time when a significant increase of testosterone from baseline was detected, always occurred during the stimulatory photoperiod, at a mean age of 256 days in Autumn born and 341 days in Spring born colts (Brown-Douglas et al., 2004). This is close to the age of 14 months at which the late spring born donkeys in the present study showed the rise in testosterone and the mounting behavior, however this could not be considered the age of puberty as no spermatozoa were present in the ejaculates.
Similar to what was observed in horses (Nishikawa, 1959) and bulls (Curtis and Amann, 1981), donkey foals’ testes showed a non-linear growth. This, however, differed from 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228
the linear testicular growth between 12 and 22 months described in colts by others (Naden et al., 1990). At 24 months, testicular width of the donkeys in this study (44.0±7.3 mm) had not yet reached that described for adult donkeys (51-59 mm; (ElWhishy, 1974; Gastal et al., 1997; Carluccio et al., 2013), thus at 2 years of age testicular growth was most probably not complete.
Data from Doppler evaluations of the jack affected with bilateral hydrocele were excluded from the results as, although this was not confirmed in stallions (Pozor and McDonnel, 2004), this condition is known to alter testicular blood flow in humans (Dudea et al., 2010). In the literature there are no data on testicular artery blood flow in prepubertal donkeys or horses and thus changes occurring around puberty have never been described. In our small group of donkeys, the change in EDV occurring around puberty was obvious, with a very low diastolic flux up to 14 months and a significant increase at 22 months, when testes were functional in all animals. Consequently at 22 and 24 months, PI and RI dropped to the lowest values recorded in the study. Similarly, a significant decrease in RI was reported to occur in children in the post-pubertal period compared to pre-puberty, when diastolic flux was usually absent (Paltiel et al., 1994). This was similar to what was observed in the young donkeys of the present study. The values for PI and RI observed at 24 months are comparable to those described in the testicular artery of 3-5 year old donkeys (Zelli et al., 2012).
Testosterone concentration was at basal levels (≤0.2 ng/ml) in all animals at 6-8 months and, although fluctuations and a gradual increase were observed, differed significantly only at 22 and 24 months, when all animals were puberal and serum testosterone was ≥0.8 ng/ml. A distinct peak was seen at 12-14 months, between June and August, when serum testosterone was 0.5 ng/ml or higher, but this was transient and, as mentioned earlier, not followed by sperm production. Testosterone had no clear relationship with age at which puberty was reached; at 20 months the two donkeys reaching puberty at 19 and at 22 months had a testosterone concentration of 0.3 and 1.1 ng/ml, respectively. This may be due to the 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254
sampling method, as single samples were collected every 2 months without previous stimulation and thus fluctuations in the normal secretion pattern may have affected the results. Moreover, the donkey reaching puberty at 22 months was the one affected by hydrocele and altered blood flow, conditions which may have slowed down sperm production and quality. At puberty, semen quality was low; this improved progressively toward the end of the study however still did not reach what has been previously described in this breed for sperm number, motility, morphology (Rota et al., 2008; Rota et al., 2012). Thus, although at a mean age of 20 months the donkey testes were able to produce spermatozoa, its full maturation was not yet reached.
It can thus be concluded that late spring born Amiata donkey colts reach puberty at about 20 months of age and that puberty is accompanied by changes in testicular size, testicular blood flow and serum testosterone concentration.
Acknowledgements
The study was funded by University of Pisa (Fondi di Ateneo), Regione Toscana and Ente Terre Regionali Toscane. We are grateful to Regione Toscana and Ente Terre Regionali Toscane for allowing us to employ the animals for this study, and to Corinne Laclie for hormonal assays.
Appendix A. Supplementary data
Supplementary data to this article are found in a supplementary file.
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Table 1
Systolic peak velocity (SPV), end dyastolic velocity (EDV), mean velocity (MV), pulsatility index (PI) and resistivity index (RI) in the supratesticular position of the testicular arteries of 4 donkeys from 8 to 24 months of age.
Age (months) / months of evaluation SPV Mean ± SD EDV Median (IQR) MV Median (IQR) PI Median (IQR) RI Median (IQR) 8 / Jan-Feb 10.1±5.1A 0.7 (1.5) 3.1 (1.3)A 3.3 (0.7) 0.95 (0.13) 10 / Mar-Apr 11.3±1.3A 0.0 (0.0)A 3.2 (1.17)A 3.4 (0.4) 1.00 (0.00) 12./ May-Jun 14.8±1.8A,a 0.0 (0.1)A 3.9 (1.5) 4.3 (1.1) 1.07 (0.03)A 14 / Jul-Aug 20.7±1.8b 0.1 (0.6) 4.7 (1.6) 4.8 (1.8)A 1.08 (0.16)A 16 / Sep-Oct 17.4±2.2A 0.3 (0.9) 4.9 (1.8) 3.6 (0.6) 0.99 (0.08) 18 / Nov-Dec 17.6±3.6A 2.1 (1.8) 6.3 (2.1) 2.3 (1.0) 0.88 (0.11) 20 / Jan-Feb 21.2±2.1 1.56 (1.41) 7.3 (1.5) 2.9 (0.3) 0.92 (0.06) 22 / Mar-Apr 26.0±3.9 5.1 (0.9) 10.8 (2.1) 1.9 (0.1) 0.79 (0.01) 24./ May-Jun 30.0±1.7B 6.5 (1.2)B 13.4 (1.5)B 1.7 (0.2)B 0.77 (0.02)B Test and P-value
ANOVA Friedman’s Friedman’s Friedman’s Friedman’s P=0.001 R2=0.8732 P<0.001 P<0.001 P<0.01 P<0.01 AB, ab (P<0.05) 339 340 341 342 343 344 345
Table 2
Age and date of the collection of the first ejaculate, the presence of the first spermatozoa and puberty in five donkeys.
Donkey First ejaculate collection Days (months) / date
First spermatozoa Days (months) / date
Puberty Days (months) / date 1 398 (13)/ July 20th 535 (18) / December 4th 570 (19) / January 8th 2 401 (13) / August 9th 553 (18) / January 8th 581 (19) / February 5th 3 422 (14) / August 9th 574 (19) / January 8th 602 (20) / February 5th 4 470 (15) / August 30th 601 (20) / January 8th 659 (22) / March 7th 5 617 (20) / February 5th 617 (20) / February 5th 617 (20) / February 5th
Mean values 461.6 (15.2) 576.0 (18.9) 605.8 (19.9) 346 347 348 349 350
FIGURE LEGENDS
Fig. 1. Testicular width (median and interquartile range) measured by ultrasound in 5 donkeys between 6 and 24 months of age (ab, cd: P<0.05).
Fig. 2. Spectral wave of the testicular artery blood flow in a donkey at 12 (left) and 24 (right) months. Observe the absence of diastolic blood flow and EDV = 0 at 12 month (pre-pubertal male).
Fig. 3. Serum testosterone concentrations in 5 donkeys between 6 and 24 months of age. ab (P<0.05). 351 352 353 354 355 356 357 358 359
