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Development of macroscopic and microscopic characteristics of ejaculates from Chios, Serres and Karaguniki breed lambs
THERIOGENOLOGY
DEVELOPMENT OF MACROSCOPIC AND MICROSCOPIC CHARACTERISTICS OF EJACULATES FROM CHIOS,SERRES AND KARAGUNIKI BREED LAMBS K. Alexopoulos> A. Karagiannidisl and P. Tsakalofr 1Clinic of Obstetrics and AI, School of Veterinarv Medicine Aristotelian University of Thessalonik; Thessaloniki, Greece Received for publication: January 25, 1991 Accepted: August 5, 1991
ABSTRACT The developmental .___. _ characteristics in the volume of the . _. ejaculate, motility (percentage of motile spermatozoa) and grade of motility (vitality), density, total number of spermatozoa per ejaculate and percentage of dead (stained) and of morphologically abnormal spermatozoa were studied in 14-to 46week-old lambs of the Chios (n=lO), Serres (~10) and Karaguniki (n=lO) breeds, born in October 1984. The first appearance of spermatozoa in the ejaculate of the Chios, Serres and Karaguniki lambs occurred at 2O.lkO.31 (THEM), 20.4iO.37 and 20.620.54 weeks of age, respectively, when the lambs had attained a body weight of 36.4&, 36.5f 0.70 and 34.9+0.99 Kg, respectively. The volume of the ejaculate, the motility and the grade of motility of spermatozoa increased at a rapid rate up to the age of 32 weeks, when the relevant values were the same as those found in the adult animal. Density of the semen and the total number of spermatozoa per ejaculate increased at a slower rate up to the age of 46 weeks, while the percentages of dead (stained) and of morphologically abnormal spermatozoa decreased significantly between 20 and 32 weeks of age. It is concluded that the quality of the semen at the time when the first spermatozoa appear in the ejaculate is not satisfactory, but it improves in the course of the ensuing 2 to 3 months. The optimal age at which the lambs may be used for artificial insemination are 32, 36 and 34 weeks for the Chios, Serres and Karaguniki breeds, respectively. Key
words : Semen, lambs, puberty
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INTRODUCTION The sexual behaviour and semen production of ram lambs are influenced by several factors, such as individual characteristics (breed, age), environment (photoperiod, temperature) and management (nutrition,social environment), which result in a large variation throughout their reproductive ifetime (l-9). The study of the development of macroscopic and microscopic characteristics of the ejaculate of lambs has been mainly based primarily on the characteristics of semen collected by means of the artificial vagina (7, 9-11) from lambs aged 6 months or older. In 1968, Skinner and Rowson (1) collected semen by electroejaculation from 42-day-old lambs, long before the animals had attained reproductive maturity. D$rmundsson and Lees (12) collected semen by electroejaculation for the purpose of determining the age at which the first spermatozoa appear in the ejaculate. Although the time at which spermatozoa appear in the ejaculate of male lambs has been investigated in several breeds, little is known about the pattern of reproductive development of indigenous sheep breeds in Greece. The object of the present study was to examine the parameters of semen collected by an electroejaculator from 12- to 14-week-old lambs of three native Greek breeds (Chios, Serres and Karaguniki). MATERIALS
AND METHODS
A total of 30 lambs (10 of the Chios.10 of the Serres and 10 of the Karaguniki breeds) were used. These animals were born in the first 10 days of October 1984 to selected ewes, inseminated after they had been synchronized for estrus. The lambs had been weaned at the age of 8 weeks and were penned all together in a shed of the Institute of Reproduction and AI, Thessaloniki, Greece ( latitude 40~ 41' N, longitude 220 48' E) in the same conditions of management and nutrition. The metereological parameters during the period of this study are shown in figure 1. Semen was collected by electrical stimulation at weekly for small ruminants intervals, using an electroejaculator (Alfred Cox Ltd), with a bipolar electrode delivering 8 V AC. These attempts started at the age of 8 weeks and were terminated at the age of 46 weeks. Immediately after collection, the quality was assessed on the following parameters :
of ejaculates
Volume. The volume of the ejaculate (ml) was determined ectly from the calibrated collection tube.
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Meteorological data
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Figure 1. The most important meteorological data. Motility. The percentage of motile spermatozoa was estimated on a microscope stage incubator at 37'C to 40°C under high power ( 400 X). Grade of motility. The rate of movement was estimated microscopically on the following arbitary scale :
o= All spermarozoa are immotile.
l= All spermatozoa show local weak wave like or circular movements. 2= Fewer than 50% of the spermatozoa show moderate progressive movement. 3= 50-80% of the spermatozoa show rapid progressive movement. 4= 90% of the spermatozoa show rapid progressive movement. 5= All spermatozoa show rapid progressive movement.
Density and total soerm number. The sperm concentration in the ejaculate measured in log/ml was estimated under a microscope equipped with a haemocytometer slide. The total sperm number per ejaculate ( Xl09 ) was calculated by multiplying density by volume of the ejaculate. Live/dead ratio and morphology of spermatozoa. The percentages of Live (unstained),dead (stained) and morphologically abnormal spermatozoa were determined from semen smears stained with nigrosin-eosin by the technique described by Dott (16).
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RESULTS The first spermatozoa appeared in the ejaculate of the lambs of all the three breeds studied at the age of 20-21 weeks, when the lambs had reached a body weight of 35 Kgs, approximately (Table 1). Comparison both of age and of the corresponding body weight of the lambs of all the three breeds did not give any statistically significant differences. Table 1. Age and body weight (it+SEM) of lambs when first spermatozoa appeared in the ejaculate Breed
Age (Weeks)
Body weight (Kgs)
20.11tO.31 20.4~tO.37 20.6~tO.54
36.4i0.57 36.520.70 34.9f0.99
Chios (n=lO> Serres (n=lO) Karaguniki (JO)
The spermatozoa appearing in the ejaculates for the first time were dead (immotile) to a very high percentage. This is shown in figure 2 diagrammatically, in which are shown the mean values of motility of spermatozoa of all the three breeds studied.
Sperm motility
20
aa
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as
si Age
OP
Figure 2. Relationship between age of lambs.
670
34
acl
40
48
44
40
lambs(weeks)
motility of spermatozoa
and
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THERIOGENOLOGY
As shown in the above figure, from the age of 20 weeks and for a period of 1 month, the motility of spermatozoa increased at a very high rate; at the age of 24 weeks it reached a considerably high level (52.8% for the lambs of Chios, 46.0% for those of Serres and 55.8% for those of Karaguniki breed). Increase in the motility rate continued at the following stages, but at a lower rate. Thus, at the age of 32 weeks were observed the highest values in all three breeds. From that age onwards till the 46th week, only small fluctuations were noted in the motility of the spermatozoa. These fluctuations were wider in the lambs of Serres breed, among whom the mean motility values found both in all semen collections and in the measurements carried out at fortnightly intervals, were significantly lower, in comparison with those of the lambs of the other two breeds (P < 0.05-P < 0.001). In Figure 3 are given the mean values of the grade of motility of spermatozoa of the lambs of all the three breeds studied.
Grade of sperm motility
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3. Grade of motility lambs.
in
relation
to the age
of
As shown therein, the few motile spermatozoa appearing. in the ejaculate for the first time had a very limited grade of motility (0.97 for the Chios, 0.80 for the Serres and 1,52 for
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the Karaguniki breed lambs). However, from the age of 20 weeks onwards and for a period of time of 2 months, the grade of motility increased at a very rapid rate. Thus, the grade of motility approached its highest values at the age of 32 weeks and subsequently remained constant up to the 46th week. However, the spermatozoa of the Serres and Karaguniki breed lambs showed a significant temporary reduction in their grade of motility at the age of 34 and 40 weeks, respectively. Among the Serres lambs, on the other hand, the mean value of the grade of motility derived from all semen collections was significantly lower in comparison with that of the two other breeds (P < 0.01). This was due to the lower grade of motility of the spermatozoa of these lambs, from the time of their appearance in the ejaculate and for the interval of the three ensuing months (P < 0.05 -P < 0.001). The number of spermatozoa present in 1 ml of semen was very low at the age of 20 weeks, as shown in figure 4, in the diagram of which are given the mean density values of the semen of lambs of all the three breeds studied. In this figure is also shown that the density of the semen of the lambs of all the three breeds, from the 20th week onwards,increased and
Density of semen
80aa24ab;Mso aa34sllJo40ta 44M Apeoflambs(weeks)
Figure 4.
Density lambs.
of semen
in relation
to
the age
of
approached its maximum values at the 44 weeks of age and remained constant for the remaining two weeks of the experi-
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ment. Naturally, this rate of increase was not the same for the three breeds: in the Serres lambs the increase in density, starting from values lower than those of the other two breeds, proceeded at a fairly rapid rate, at least during the first few weeks after the 20th week, in comparison with the density of semen of the lambs of the other two breeds. However, the mean value of the density of semen derived from all semen collections of the Serres iambs was lower than that of the lambs of the other two breeds (P < 0.01); likewise, in almost all measurements made at fortnightly intervals, the Serres lambs were inferior to the lambs of the two other breeds as regards the values of density of semen (P < 0.05 P < 0.001). The ejaculates, in which spermatozoa first started to appear, contained a very small number of spermatozoa, both per 1 ml, as well as in the whole of the ejaculate. In the diagram of figure 5 are given the mean values of spermatozoa per ejacqlate among the lambs of all the three breeds studied.
Spermatozoa/ejaculate
Age of Iambs (weeks)
Figure 5.
Number of spermatozoa to the age of lambs.
per ejaculate
in relation
From this figure, it appears that the number of spermatozoa per ejaculate starting to appear from the 20th week, increased at a relatively slow rate, up to the age of 38 weeks, when it was accelerated, till the 44th week. From that age onwards, the number of spermatozoa per ejaculate remained
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constant, till the end of the observation period in the lambs of all the three breeds. The differences observed between breeds as regards the mean number of spermatozoa per ejaculate derived from all semen collections were proportionate to those reported for the density of semen. In figure 6 are given the mean values of the percentage of dead (stained) spermatozoa of the lambs of all the three breeds of the experimental animals.
Dead spermatozoa m lpIomataaa WI 100 w 60 40
0 20I8420aesOi3a84sl3~40424448 Age
oflambs(weeks)
Figure 6. Percentage of dead (stained) spermatozoa relation to the age of lambs
in
Prom this, it becomes obvious that the percentage of dead spermatozoa was very high at the age of 20 weeks 81,5% for the Chios, 83.5% for the Serres and 76.3% for the Karaguniki breed lambs). From this age onwards (particularly during the following month) the percentage of dead spermatozoa diminished at a very rapid rate, which however was subsequently retarded till the 32nd week, when the percentage of dead spermatozoa reached its lowest values; from then onwards it showed small fluctuations. The latter were wider in the Serres lambs, among whom the mean value of the percentage of dead spermatozoa derived both from all semen collections and almost all measurements made at fortnightly intervals, was significantly higher, in relation to those of the two other breeds (P < 0.05 - P < 0.001).
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THERIOGENOLOGY
The first spermatozoa in the ejaculate of the lambs showed morphological abnormalities to a very high percentage (76.0% among Chios, 86.1% among Serres and 85.3% among the Karaguniki breed lambs). Figure 7 shows that the percentage of morphologically abnormal spermatozoa was diminished from the age of 20 weeks onwards for all the breeds studied.
Abnormal spermatozoa
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Ageoflambs (weeks)
Figure 7.
I ?ercentage
of abnormal to the age of lambs.
spermatozoa
in
relation
Tn the case of Serres and Karaguniki breed lambs the percentage of morphologically abnormal spermatozoa diminished till the 36th week; from then onwards it remained constant, till the end of the observation period. Among the Chios lambs this stabilization was observed from the age of 32 weeks. The rate of reduction in the percentage of the morphologically abnormal spermatozoa was not the same in the three breeds; the reduction of this percentage proceeded at a relatively slow rate among the Serres lambs, at a more rapid rate among Karaguniki lambs and at a still more rapid rate among the Chios lambs. The percantage of morphologically abnormal spermatozoa among Serres lambs, in comparison with that of the two other breeds, was significantly higher as regards both the mean value derived from all semen collections (P < 0.001) and most of the fortnightly measurements (P < 0.05 - P < 0.001). Significant differences between breeds were observed only during the last two months of observations.
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THERIOGENOLOGY DISCUSSION
The time of onset of puberty and the age at which the first spermatozoa appear in the ejaculate varies among breeds (1, 10, 14-17) and within breeds. Owing to the duration of the cellular processes (18) and cellular degenerations, which occur in the first spermatogenic cycles (19), 2 to 3 months are required for spermatogenesis to be completed and for the release of the first sperm cells. Since two more weeks are necessary for epididymal transit (20,21), the first spermatozoa can be collected around the 5th to the 6th month of age in Ile-de-France, PrCalpes and Romanov male lambs (5, 16). In our study the first spermatozoa appeared in the ejaculate of lambs of all the three Greek native breeds at the age of 20 weeks (about 5 months), when the live weight ranged from 35 to 37 Kg. Dy'rmundsson (4) in a review concluded that body weight is a better criterion for the attainment of puberty than the chronological age alone. The quality of semen was not good at the age of 20 weeks, i.e. when the first spermatozoa appeared in the ejaculate, but it improved rapidly in the course of the following 2-3 months. The low quality of semen in the first months of reproductive life has been observed in several breeds : Dorper (lo), Suffolk (l), Prealpes (16) and Aragonesa (9). The above conclusionwas reached on the basis of the examination of certain microscopic characteristics of the ejaculate, which, according to Mann and Lutwak-Mann (22) constitute the commonest criteria used for semen quality evaluation. These characteristics are motility (percentage of motile) and the grade of motility (vitality) of spermatozoa, density and/or total number of spermatozoa per ejaculate, and the percentages of dead (stained) and of morphologically abnormal spermatozoa. These parameters in the semen of male lambs were also studied by other workers (7, 9, 10, 11). The collection of semen was made by means of the artificial vagina and, therefore, their data were collected from lambs at the age of 6-7 months, while the data of the present study concern younger lambs (20 weeks old). The values of motility and the grade of motility of the first spermatozoa found in this study and by the above workers were low and were attributed, by most of them, to the considerably large numbers of morphologically abnormal spermatozoa present in the ejaculate. The large number of morphologically abnormal spermatozoa at this age should be due to the immaturity of the seminal epithelium and the degenerative phenomena in the cells which occur during the first spermatogenic cycles (19) and result in an imperfect formation of spermatozoa (high proportion of primary abnormalities). As shown by the results of this study, motility and the grade of motility of spermatozoa increased at a very rapid rate till the age of 24 weeks; this finding is in agreement
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with those of Folch (9) and Lauda et al. (11). It is a general feature in all ram lambs, but the evolution to better quality varies with breeds : it is fairly rapid (1 month) in Prialpes and slower (3 months) in Ile-de-France lambs. It should be pointed out that in our study these characteristics were stabilized relatively fast (i.e. about the 32nd week of life). However, between the 32nd and 43th week, a time interval that coincided with the summer months, a considerable drop in the values of motility and the grade of motility in the semen of the lambs of all breeds was observed. It appears that the high ambient temperature exerted a negative influence on the testes of the lambs, as this has been shown to be the case among rams by Stamatiadis (23). The unfavourable effect of high temperature on testicular activity of rams has been demonstrated in experimental conditions (24), but the mechanisms responsible for the effect of heat on the testis are not well understood (25). Under natural conditions there is lack of uniformity in the influence of heat on semen production, because there are important breed differences in sensitivity to heat (26). On the other hand there are other climatic factors that may favour the action of thermoregulating mechanisms, which would counteract the effect of heat (27-31). motility and the grade of motility of sperApart from matozoa, the first ejaculates in which spermatozoa first appeared, contained a very small number of spermatozoa, both per ml and per ejaculate. Similar results were reported by other workers (7, 10, 3$). The small number of spermatozoa contained in the ejaculate of lambs must be due, on one hand, to the fact that in each seminal tubule only a small number of spermatogonia is activated and on, the other hand, to the fact that spermatogenesis does not start simultaneously in all seminal tubules of the same testis. Eventually, the number of spermatozoa produced by the testes was increased markedly. Thus, the reproductive potential of ram lambs was likely to rise at a steady rate with advancing age and body development during the period from puberty to full adulthood (1). REFERENCES 1.
Skinner, J. D. and Rowson, and cross-bred ram lambs. 488 (1968).
2.
Skinner, J. D., Booth, W. D., Rowson, L. E. A. and The post-natal development of the Karg, H. reproductive tract of the Suffolk ram and changes in the gonadotrophin content of the pituitary. J. Reprod. Fert. 16: 463-477 (1968).
OCTOBER 1991 VOL. 36 NO. 4
L. E. A. Puberty in Suffolk J. Reprod. Pert., 16: 479-
677
THERIOGENOLOGY 3.
Land, R. B. The mating behaviour and semen characteristics Scottish of Finnish Landrace and Blackface rams. Anim. Prod., 12: 551-560 (1970).
4.
Dyrmundsson, 6. R. Puberty and early reproductive perAnim. Breed. Abs., formance in sheep. II. Ram Lambs. 41: 9 (1973).
5.
Colas, G., Personnic,D., Courot, M. et Ortavant, R. Influence de rythme de recolte sur la production de spermatozoides le jeune b&lier romanov. Annls chez Biol. anim. Bioch. Biophys. 24: 189-198 (1975).
6.
Testis growth and plasma Land, R. B. and Carr, W. R. LH concentration following hemicastration and its relation to female prolificacy in sheep. J. Reprod., Fert. 45: 495-501 (1975).
7.
photoperiodism Alberio, R. and Colas, G. Influence of of the young Ile-de-France on the sexual development ram. Congress Animal VIIIth International Artificial InseminatioOnlf Crocow. Production and (1976).
8.
Land, R. B. Genetic variation and improvement. In Reproduction 3rd ed., ( ed. H. H. in Domestic Animals, Cole and P. T. Cupps), pp 577-604. Academic Press, New York (1970).
9.
photoperiodism and Influence of age, Folch, J. temperature on semen production of rams. Proc. of EEC Seminar on Animal Production, Nouzilly, France 6-7 Oct. 1983, pp 141-159.
10.
Puberty in the male Louw, D. F. J. and Joubert, D.M. S. Afric. Agric. Sci. z: Dorper sheep and Boer goat. 509-520 (1964) (Anim. Breed. Abstr., 431, 1965).
11.
stoic, L., Krizek, J. and Louda, F., Doney, J. M., of sexual activity and J. The development Smerha, of the prolific two semen production in ram lambs Anim. Prod. 33: breeds: Romanov and Finnish Landrace. 143-148 (1981).
12.
Dyrmundsson, 6. R. and Lees, J. Puberal development Clun-Forest ram lambs in relation to time of birth. Agric. Sci., UK, 79: 83-89 (1972).
13.
staining of spermatozoa in the Dott, Ii. Partial Proc. 3rd Int. Congr. Anim. nigrosin-eosin stain. Reprod., Cambridge, sec. 3: 42-43 (1956).
678
of J.
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14.
Watson, R. II., Sapsford, C. S. and McCance, I. The development of the testis, epididymis and penis in the young Merino ram. Aust. J. agric. Res. 1: 774-590 (1956).
15.
of the testes Barr, A. M. The postnatal development and epididymidis in the Awassi rams under Lebanese environment. Magon Puplications, Serie Scientitique, Lebanon No 30 (1969).
16.
Production spermaColas, G. et Zinsner-Pflimlin, F. tique et croisance testiculaire chez l'agneau de race Journdes de la Recherche Ile-de-France et PrCalpes. Ovine et Caprine en France (1975). Ed. ITOVIC- SPEOC, Paris), pp. 162-185.
17.
Madhat, M. N. El-Shobokshy, Ghannam, S. A. M., and in Awassi ram lambs. I. Effect of inA.S. Puberty on the onset creasing protein level of sexual maturity. Z. Tierzuchtg. Zuchtgsbiol. 94: 226-234 (1978).
18.
Ortavan, R. Le cycle spermatogdnitique chez le be'lier. Thise Dr. Bs Sci., Paris. CNRS A 3118 (1958).
19.
du testicule chez l'agneau. Courot, M. D6veloppement de la spermatogbn&e. Ann. Biol. Anim. Etablissement Biochim. Biophys., 2: 25-42 (1962).
20.
Influence de la frequence des Amir, D. et Ortavan, R. collect-es sur la duree du transit des spermatozoides An. Biol. anim. dans le canal Cpididymaire du bilier. Bioch. Bioph. 8: 195-207 (1968).
21.
Courot, M., Hochereau, M.T., Ortavan, R. Spermatogenesis, In: Johnson, A. D., Gomes, W.R. and VanDemark, N. I. (Ed.). The testis, New York: Academic Press. 1, 339-342 (1970).
22.
Mann, T. and Lutwak-Mann, C. Male Reproductive Function and Semen. Springer-Verlag, Berlin-Heidelberg, New York. (1981).
23.
Stamatiadis, K. Seasonal variation in semen production of Chios and Fiesian rams. Dissertation. Aristotelian University of Thessaloniki, Greece (1982).
24.
Waites, G. M. H. et O&avant, R. Effect pre'coces d'une br&ve &valuation de la tempdrature testiculaire sur la spermatogdndse du b6lier. An. Biol. Anim. Bioch. Bioph. 8, 323-331 (1968).
25.
(Ed. C. A. Setchel. B. P. In: The Mammalian Testis, Finn). (Elek Books Limited, London), pp 90-113 (1978).
OCTOBER 1991 VOL. 36 NO. 4
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THERIOGENOLOGY
26.
Lindsay, D. R. Sexual activity and semen production at high temperatures. J. Reprod. Fe&.,=: l-8 (1969).
27.
Sachni, K. L. and Roy, A. A note on seasonal variation in the occurance of abnormal spermatozoa in different under tropical conditions. breeds of sheep and goat Indian J. Anim. Sci., 42_: 501-504 (1972).
26.
Aboul-Naga, A. Galal, E. S. E., El-Gamal, A. A., and El-Fouly, M. A. Male reproductive characteristics of Merino and Ossini sheep and their crosses. Anim. Prod. 27: 261-267 (1978).
29.
Mittal, J.P. and Ghosh, P. K. Comparative semen charJaisalmeri of Corriedale, Marwari and acteristics rams maintained under hot arid conditions. J. Aqri. Sci., Cam. 2: l-4 (1979).
30.
Variazioni and Branca, A. Manunta, G. Cappai, P., dell'eiaculato . caratteristiche stagionali delle Frisona't arieti di razza Sarda, Romanov e mettici Sarda. IV Cong. della sot. Italiana di Patologia di allevamento degli ovini e dei caprine. Alghero (1981).
31.
l'activitd sexFolch, J. et Roca, R. Importance de du mile dans le d&eloppement du croisement uelle Options Miditerraniennes. Vol. industriel en Espagne. III, 135-141 (Ed. C. I. H. E. A. M Paris) (1981).
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DEVELOPMENT OF MACROSCOPIC AND MICROSCOPIC CHARACTERISTICS OF EJACULATES FROM CHIOS,SERRES AND KARAGUNIKI BREED LAMBS K. Alexopoulos> A. Karagiannidisl and P. Tsakalofr 1Clinic of Obstetrics and AI, School of Veterinarv Medicine Aristotelian University of Thessalonik; Thessaloniki, Greece Received for publication: January 25, 1991 Accepted: August 5, 1991
ABSTRACT The developmental .___. _ characteristics in the volume of the . _. ejaculate, motility (percentage of motile spermatozoa) and grade of motility (vitality), density, total number of spermatozoa per ejaculate and percentage of dead (stained) and of morphologically abnormal spermatozoa were studied in 14-to 46week-old lambs of the Chios (n=lO), Serres (~10) and Karaguniki (n=lO) breeds, born in October 1984. The first appearance of spermatozoa in the ejaculate of the Chios, Serres and Karaguniki lambs occurred at 2O.lkO.31 (THEM), 20.4iO.37 and 20.620.54 weeks of age, respectively, when the lambs had attained a body weight of 36.4&, 36.5f 0.70 and 34.9+0.99 Kg, respectively. The volume of the ejaculate, the motility and the grade of motility of spermatozoa increased at a rapid rate up to the age of 32 weeks, when the relevant values were the same as those found in the adult animal. Density of the semen and the total number of spermatozoa per ejaculate increased at a slower rate up to the age of 46 weeks, while the percentages of dead (stained) and of morphologically abnormal spermatozoa decreased significantly between 20 and 32 weeks of age. It is concluded that the quality of the semen at the time when the first spermatozoa appear in the ejaculate is not satisfactory, but it improves in the course of the ensuing 2 to 3 months. The optimal age at which the lambs may be used for artificial insemination are 32, 36 and 34 weeks for the Chios, Serres and Karaguniki breeds, respectively. Key
words : Semen, lambs, puberty
OCTOBER 1991 VOL. 36 NO. 4
THERIOGENOLOGY
INTRODUCTION The sexual behaviour and semen production of ram lambs are influenced by several factors, such as individual characteristics (breed, age), environment (photoperiod, temperature) and management (nutrition,social environment), which result in a large variation throughout their reproductive ifetime (l-9). The study of the development of macroscopic and microscopic characteristics of the ejaculate of lambs has been mainly based primarily on the characteristics of semen collected by means of the artificial vagina (7, 9-11) from lambs aged 6 months or older. In 1968, Skinner and Rowson (1) collected semen by electroejaculation from 42-day-old lambs, long before the animals had attained reproductive maturity. D$rmundsson and Lees (12) collected semen by electroejaculation for the purpose of determining the age at which the first spermatozoa appear in the ejaculate. Although the time at which spermatozoa appear in the ejaculate of male lambs has been investigated in several breeds, little is known about the pattern of reproductive development of indigenous sheep breeds in Greece. The object of the present study was to examine the parameters of semen collected by an electroejaculator from 12- to 14-week-old lambs of three native Greek breeds (Chios, Serres and Karaguniki). MATERIALS
AND METHODS
A total of 30 lambs (10 of the Chios.10 of the Serres and 10 of the Karaguniki breeds) were used. These animals were born in the first 10 days of October 1984 to selected ewes, inseminated after they had been synchronized for estrus. The lambs had been weaned at the age of 8 weeks and were penned all together in a shed of the Institute of Reproduction and AI, Thessaloniki, Greece ( latitude 40~ 41' N, longitude 220 48' E) in the same conditions of management and nutrition. The metereological parameters during the period of this study are shown in figure 1. Semen was collected by electrical stimulation at weekly for small ruminants intervals, using an electroejaculator (Alfred Cox Ltd), with a bipolar electrode delivering 8 V AC. These attempts started at the age of 8 weeks and were terminated at the age of 46 weeks. Immediately after collection, the quality was assessed on the following parameters :
of ejaculates
Volume. The volume of the ejaculate (ml) was determined ectly from the calibrated collection tube.
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OCTOBER 1991 VOL. 36 NO. 4
THERIOGENOLOGY
Meteorological data
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00
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am. au.
Feb.
me?.Apr.
nQ
h.
&IL
Au&
3
Fortnight
Figure 1. The most important meteorological data. Motility. The percentage of motile spermatozoa was estimated on a microscope stage incubator at 37'C to 40°C under high power ( 400 X). Grade of motility. The rate of movement was estimated microscopically on the following arbitary scale :
o= All spermarozoa are immotile.
l= All spermatozoa show local weak wave like or circular movements. 2= Fewer than 50% of the spermatozoa show moderate progressive movement. 3= 50-80% of the spermatozoa show rapid progressive movement. 4= 90% of the spermatozoa show rapid progressive movement. 5= All spermatozoa show rapid progressive movement.
Density and total soerm number. The sperm concentration in the ejaculate measured in log/ml was estimated under a microscope equipped with a haemocytometer slide. The total sperm number per ejaculate ( Xl09 ) was calculated by multiplying density by volume of the ejaculate. Live/dead ratio and morphology of spermatozoa. The percentages of Live (unstained),dead (stained) and morphologically abnormal spermatozoa were determined from semen smears stained with nigrosin-eosin by the technique described by Dott (16).
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RESULTS The first spermatozoa appeared in the ejaculate of the lambs of all the three breeds studied at the age of 20-21 weeks, when the lambs had reached a body weight of 35 Kgs, approximately (Table 1). Comparison both of age and of the corresponding body weight of the lambs of all the three breeds did not give any statistically significant differences. Table 1. Age and body weight (it+SEM) of lambs when first spermatozoa appeared in the ejaculate Breed
Age (Weeks)
Body weight (Kgs)
20.11tO.31 20.4~tO.37 20.6~tO.54
36.4i0.57 36.520.70 34.9f0.99
Chios (n=lO> Serres (n=lO) Karaguniki (JO)
The spermatozoa appearing in the ejaculates for the first time were dead (immotile) to a very high percentage. This is shown in figure 2 diagrammatically, in which are shown the mean values of motility of spermatozoa of all the three breeds studied.
Sperm motility
20
aa
a4
as
si Age
OP
Figure 2. Relationship between age of lambs.
670
34
acl
40
48
44
40
lambs(weeks)
motility of spermatozoa
and
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THERIOGENOLOGY
As shown in the above figure, from the age of 20 weeks and for a period of 1 month, the motility of spermatozoa increased at a very high rate; at the age of 24 weeks it reached a considerably high level (52.8% for the lambs of Chios, 46.0% for those of Serres and 55.8% for those of Karaguniki breed). Increase in the motility rate continued at the following stages, but at a lower rate. Thus, at the age of 32 weeks were observed the highest values in all three breeds. From that age onwards till the 46th week, only small fluctuations were noted in the motility of the spermatozoa. These fluctuations were wider in the lambs of Serres breed, among whom the mean motility values found both in all semen collections and in the measurements carried out at fortnightly intervals, were significantly lower, in comparison with those of the lambs of the other two breeds (P < 0.05-P < 0.001). In Figure 3 are given the mean values of the grade of motility of spermatozoa of the lambs of all the three breeds studied.
Grade of sperm motility
1-A.Euqdblbmd 1 0 ab
’
as
’
24
’
80
’
aa
’
a0
’
aa
’
34
’
sb
*
sll4o
’
’
ra
’
44
’
48
he oflambs(weeks)
Figure
3. Grade of motility lambs.
in
relation
to the age
of
As shown therein, the few motile spermatozoa appearing. in the ejaculate for the first time had a very limited grade of motility (0.97 for the Chios, 0.80 for the Serres and 1,52 for
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the Karaguniki breed lambs). However, from the age of 20 weeks onwards and for a period of time of 2 months, the grade of motility increased at a very rapid rate. Thus, the grade of motility approached its highest values at the age of 32 weeks and subsequently remained constant up to the 46th week. However, the spermatozoa of the Serres and Karaguniki breed lambs showed a significant temporary reduction in their grade of motility at the age of 34 and 40 weeks, respectively. Among the Serres lambs, on the other hand, the mean value of the grade of motility derived from all semen collections was significantly lower in comparison with that of the two other breeds (P < 0.01). This was due to the lower grade of motility of the spermatozoa of these lambs, from the time of their appearance in the ejaculate and for the interval of the three ensuing months (P < 0.05 -P < 0.001). The number of spermatozoa present in 1 ml of semen was very low at the age of 20 weeks, as shown in figure 4, in the diagram of which are given the mean density values of the semen of lambs of all the three breeds studied. In this figure is also shown that the density of the semen of the lambs of all the three breeds, from the 20th week onwards,increased and
Density of semen
80aa24ab;Mso aa34sllJo40ta 44M Apeoflambs(weeks)
Figure 4.
Density lambs.
of semen
in relation
to
the age
of
approached its maximum values at the 44 weeks of age and remained constant for the remaining two weeks of the experi-
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THERIOGENOLOGY
ment. Naturally, this rate of increase was not the same for the three breeds: in the Serres lambs the increase in density, starting from values lower than those of the other two breeds, proceeded at a fairly rapid rate, at least during the first few weeks after the 20th week, in comparison with the density of semen of the lambs of the other two breeds. However, the mean value of the density of semen derived from all semen collections of the Serres iambs was lower than that of the lambs of the other two breeds (P < 0.01); likewise, in almost all measurements made at fortnightly intervals, the Serres lambs were inferior to the lambs of the two other breeds as regards the values of density of semen (P < 0.05 P < 0.001). The ejaculates, in which spermatozoa first started to appear, contained a very small number of spermatozoa, both per 1 ml, as well as in the whole of the ejaculate. In the diagram of figure 5 are given the mean values of spermatozoa per ejacqlate among the lambs of all the three breeds studied.
Spermatozoa/ejaculate
Age of Iambs (weeks)
Figure 5.
Number of spermatozoa to the age of lambs.
per ejaculate
in relation
From this figure, it appears that the number of spermatozoa per ejaculate starting to appear from the 20th week, increased at a relatively slow rate, up to the age of 38 weeks, when it was accelerated, till the 44th week. From that age onwards, the number of spermatozoa per ejaculate remained
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THERIOGENOLOGY
constant, till the end of the observation period in the lambs of all the three breeds. The differences observed between breeds as regards the mean number of spermatozoa per ejaculate derived from all semen collections were proportionate to those reported for the density of semen. In figure 6 are given the mean values of the percentage of dead (stained) spermatozoa of the lambs of all the three breeds of the experimental animals.
Dead spermatozoa m lpIomataaa WI 100 w 60 40
0 20I8420aesOi3a84sl3~40424448 Age
oflambs(weeks)
Figure 6. Percentage of dead (stained) spermatozoa relation to the age of lambs
in
Prom this, it becomes obvious that the percentage of dead spermatozoa was very high at the age of 20 weeks 81,5% for the Chios, 83.5% for the Serres and 76.3% for the Karaguniki breed lambs). From this age onwards (particularly during the following month) the percentage of dead spermatozoa diminished at a very rapid rate, which however was subsequently retarded till the 32nd week, when the percentage of dead spermatozoa reached its lowest values; from then onwards it showed small fluctuations. The latter were wider in the Serres lambs, among whom the mean value of the percentage of dead spermatozoa derived both from all semen collections and almost all measurements made at fortnightly intervals, was significantly higher, in relation to those of the two other breeds (P < 0.05 - P < 0.001).
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THERIOGENOLOGY
The first spermatozoa in the ejaculate of the lambs showed morphological abnormalities to a very high percentage (76.0% among Chios, 86.1% among Serres and 85.3% among the Karaguniki breed lambs). Figure 7 shows that the percentage of morphologically abnormal spermatozoa was diminished from the age of 20 weeks onwards for all the breeds studied.
Abnormal spermatozoa
01 “‘I ” ” w~a4a6mtwaa34es~40424440
”
’
”
Ageoflambs (weeks)
Figure 7.
I ?ercentage
of abnormal to the age of lambs.
spermatozoa
in
relation
Tn the case of Serres and Karaguniki breed lambs the percentage of morphologically abnormal spermatozoa diminished till the 36th week; from then onwards it remained constant, till the end of the observation period. Among the Chios lambs this stabilization was observed from the age of 32 weeks. The rate of reduction in the percentage of the morphologically abnormal spermatozoa was not the same in the three breeds; the reduction of this percentage proceeded at a relatively slow rate among the Serres lambs, at a more rapid rate among Karaguniki lambs and at a still more rapid rate among the Chios lambs. The percantage of morphologically abnormal spermatozoa among Serres lambs, in comparison with that of the two other breeds, was significantly higher as regards both the mean value derived from all semen collections (P < 0.001) and most of the fortnightly measurements (P < 0.05 - P < 0.001). Significant differences between breeds were observed only during the last two months of observations.
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675
THERIOGENOLOGY DISCUSSION
The time of onset of puberty and the age at which the first spermatozoa appear in the ejaculate varies among breeds (1, 10, 14-17) and within breeds. Owing to the duration of the cellular processes (18) and cellular degenerations, which occur in the first spermatogenic cycles (19), 2 to 3 months are required for spermatogenesis to be completed and for the release of the first sperm cells. Since two more weeks are necessary for epididymal transit (20,21), the first spermatozoa can be collected around the 5th to the 6th month of age in Ile-de-France, PrCalpes and Romanov male lambs (5, 16). In our study the first spermatozoa appeared in the ejaculate of lambs of all the three Greek native breeds at the age of 20 weeks (about 5 months), when the live weight ranged from 35 to 37 Kg. Dy'rmundsson (4) in a review concluded that body weight is a better criterion for the attainment of puberty than the chronological age alone. The quality of semen was not good at the age of 20 weeks, i.e. when the first spermatozoa appeared in the ejaculate, but it improved rapidly in the course of the following 2-3 months. The low quality of semen in the first months of reproductive life has been observed in several breeds : Dorper (lo), Suffolk (l), Prealpes (16) and Aragonesa (9). The above conclusionwas reached on the basis of the examination of certain microscopic characteristics of the ejaculate, which, according to Mann and Lutwak-Mann (22) constitute the commonest criteria used for semen quality evaluation. These characteristics are motility (percentage of motile) and the grade of motility (vitality) of spermatozoa, density and/or total number of spermatozoa per ejaculate, and the percentages of dead (stained) and of morphologically abnormal spermatozoa. These parameters in the semen of male lambs were also studied by other workers (7, 9, 10, 11). The collection of semen was made by means of the artificial vagina and, therefore, their data were collected from lambs at the age of 6-7 months, while the data of the present study concern younger lambs (20 weeks old). The values of motility and the grade of motility of the first spermatozoa found in this study and by the above workers were low and were attributed, by most of them, to the considerably large numbers of morphologically abnormal spermatozoa present in the ejaculate. The large number of morphologically abnormal spermatozoa at this age should be due to the immaturity of the seminal epithelium and the degenerative phenomena in the cells which occur during the first spermatogenic cycles (19) and result in an imperfect formation of spermatozoa (high proportion of primary abnormalities). As shown by the results of this study, motility and the grade of motility of spermatozoa increased at a very rapid rate till the age of 24 weeks; this finding is in agreement
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THERIOGENOLOGY
with those of Folch (9) and Lauda et al. (11). It is a general feature in all ram lambs, but the evolution to better quality varies with breeds : it is fairly rapid (1 month) in Prialpes and slower (3 months) in Ile-de-France lambs. It should be pointed out that in our study these characteristics were stabilized relatively fast (i.e. about the 32nd week of life). However, between the 32nd and 43th week, a time interval that coincided with the summer months, a considerable drop in the values of motility and the grade of motility in the semen of the lambs of all breeds was observed. It appears that the high ambient temperature exerted a negative influence on the testes of the lambs, as this has been shown to be the case among rams by Stamatiadis (23). The unfavourable effect of high temperature on testicular activity of rams has been demonstrated in experimental conditions (24), but the mechanisms responsible for the effect of heat on the testis are not well understood (25). Under natural conditions there is lack of uniformity in the influence of heat on semen production, because there are important breed differences in sensitivity to heat (26). On the other hand there are other climatic factors that may favour the action of thermoregulating mechanisms, which would counteract the effect of heat (27-31). motility and the grade of motility of sperApart from matozoa, the first ejaculates in which spermatozoa first appeared, contained a very small number of spermatozoa, both per ml and per ejaculate. Similar results were reported by other workers (7, 10, 3$). The small number of spermatozoa contained in the ejaculate of lambs must be due, on one hand, to the fact that in each seminal tubule only a small number of spermatogonia is activated and on, the other hand, to the fact that spermatogenesis does not start simultaneously in all seminal tubules of the same testis. Eventually, the number of spermatozoa produced by the testes was increased markedly. Thus, the reproductive potential of ram lambs was likely to rise at a steady rate with advancing age and body development during the period from puberty to full adulthood (1). REFERENCES 1.
Skinner, J. D. and Rowson, and cross-bred ram lambs. 488 (1968).
2.
Skinner, J. D., Booth, W. D., Rowson, L. E. A. and The post-natal development of the Karg, H. reproductive tract of the Suffolk ram and changes in the gonadotrophin content of the pituitary. J. Reprod. Fert. 16: 463-477 (1968).
OCTOBER 1991 VOL. 36 NO. 4
L. E. A. Puberty in Suffolk J. Reprod. Pert., 16: 479-
677
THERIOGENOLOGY 3.
Land, R. B. The mating behaviour and semen characteristics Scottish of Finnish Landrace and Blackface rams. Anim. Prod., 12: 551-560 (1970).
4.
Dyrmundsson, 6. R. Puberty and early reproductive perAnim. Breed. Abs., formance in sheep. II. Ram Lambs. 41: 9 (1973).
5.
Colas, G., Personnic,D., Courot, M. et Ortavant, R. Influence de rythme de recolte sur la production de spermatozoides le jeune b&lier romanov. Annls chez Biol. anim. Bioch. Biophys. 24: 189-198 (1975).
6.
Testis growth and plasma Land, R. B. and Carr, W. R. LH concentration following hemicastration and its relation to female prolificacy in sheep. J. Reprod., Fert. 45: 495-501 (1975).
7.
photoperiodism Alberio, R. and Colas, G. Influence of of the young Ile-de-France on the sexual development ram. Congress Animal VIIIth International Artificial InseminatioOnlf Crocow. Production and (1976).
8.
Land, R. B. Genetic variation and improvement. In Reproduction 3rd ed., ( ed. H. H. in Domestic Animals, Cole and P. T. Cupps), pp 577-604. Academic Press, New York (1970).
9.
photoperiodism and Influence of age, Folch, J. temperature on semen production of rams. Proc. of EEC Seminar on Animal Production, Nouzilly, France 6-7 Oct. 1983, pp 141-159.
10.
Puberty in the male Louw, D. F. J. and Joubert, D.M. S. Afric. Agric. Sci. z: Dorper sheep and Boer goat. 509-520 (1964) (Anim. Breed. Abstr., 431, 1965).
11.
stoic, L., Krizek, J. and Louda, F., Doney, J. M., of sexual activity and J. The development Smerha, of the prolific two semen production in ram lambs Anim. Prod. 33: breeds: Romanov and Finnish Landrace. 143-148 (1981).
12.
Dyrmundsson, 6. R. and Lees, J. Puberal development Clun-Forest ram lambs in relation to time of birth. Agric. Sci., UK, 79: 83-89 (1972).
13.
staining of spermatozoa in the Dott, Ii. Partial Proc. 3rd Int. Congr. Anim. nigrosin-eosin stain. Reprod., Cambridge, sec. 3: 42-43 (1956).
678
of J.
OCTOBER1991VOL.36NO.4
THERIOGENOLOGY
14.
Watson, R. II., Sapsford, C. S. and McCance, I. The development of the testis, epididymis and penis in the young Merino ram. Aust. J. agric. Res. 1: 774-590 (1956).
15.
of the testes Barr, A. M. The postnatal development and epididymidis in the Awassi rams under Lebanese environment. Magon Puplications, Serie Scientitique, Lebanon No 30 (1969).
16.
Production spermaColas, G. et Zinsner-Pflimlin, F. tique et croisance testiculaire chez l'agneau de race Journdes de la Recherche Ile-de-France et PrCalpes. Ovine et Caprine en France (1975). Ed. ITOVIC- SPEOC, Paris), pp. 162-185.
17.
Madhat, M. N. El-Shobokshy, Ghannam, S. A. M., and in Awassi ram lambs. I. Effect of inA.S. Puberty on the onset creasing protein level of sexual maturity. Z. Tierzuchtg. Zuchtgsbiol. 94: 226-234 (1978).
18.
Ortavan, R. Le cycle spermatogdnitique chez le be'lier. Thise Dr. Bs Sci., Paris. CNRS A 3118 (1958).
19.
du testicule chez l'agneau. Courot, M. D6veloppement de la spermatogbn&e. Ann. Biol. Anim. Etablissement Biochim. Biophys., 2: 25-42 (1962).
20.
Influence de la frequence des Amir, D. et Ortavan, R. collect-es sur la duree du transit des spermatozoides An. Biol. anim. dans le canal Cpididymaire du bilier. Bioch. Bioph. 8: 195-207 (1968).
21.
Courot, M., Hochereau, M.T., Ortavan, R. Spermatogenesis, In: Johnson, A. D., Gomes, W.R. and VanDemark, N. I. (Ed.). The testis, New York: Academic Press. 1, 339-342 (1970).
22.
Mann, T. and Lutwak-Mann, C. Male Reproductive Function and Semen. Springer-Verlag, Berlin-Heidelberg, New York. (1981).
23.
Stamatiadis, K. Seasonal variation in semen production of Chios and Fiesian rams. Dissertation. Aristotelian University of Thessaloniki, Greece (1982).
24.
Waites, G. M. H. et O&avant, R. Effect pre'coces d'une br&ve &valuation de la tempdrature testiculaire sur la spermatogdndse du b6lier. An. Biol. Anim. Bioch. Bioph. 8, 323-331 (1968).
25.
(Ed. C. A. Setchel. B. P. In: The Mammalian Testis, Finn). (Elek Books Limited, London), pp 90-113 (1978).
OCTOBER 1991 VOL. 36 NO. 4
679
THERIOGENOLOGY
26.
Lindsay, D. R. Sexual activity and semen production at high temperatures. J. Reprod. Fe&.,=: l-8 (1969).
27.
Sachni, K. L. and Roy, A. A note on seasonal variation in the occurance of abnormal spermatozoa in different under tropical conditions. breeds of sheep and goat Indian J. Anim. Sci., 42_: 501-504 (1972).
26.
Aboul-Naga, A. Galal, E. S. E., El-Gamal, A. A., and El-Fouly, M. A. Male reproductive characteristics of Merino and Ossini sheep and their crosses. Anim. Prod. 27: 261-267 (1978).
29.
Mittal, J.P. and Ghosh, P. K. Comparative semen charJaisalmeri of Corriedale, Marwari and acteristics rams maintained under hot arid conditions. J. Aqri. Sci., Cam. 2: l-4 (1979).
30.
Variazioni and Branca, A. Manunta, G. Cappai, P., dell'eiaculato . caratteristiche stagionali delle Frisona't arieti di razza Sarda, Romanov e mettici Sarda. IV Cong. della sot. Italiana di Patologia di allevamento degli ovini e dei caprine. Alghero (1981).
31.
l'activitd sexFolch, J. et Roca, R. Importance de du mile dans le d&eloppement du croisement uelle Options Miditerraniennes. Vol. industriel en Espagne. III, 135-141 (Ed. C. I. H. E. A. M Paris) (1981).
660
OCTOBER 1991 VOL. 36 NO. 4