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Developmental changes in testicular interstitium in the Najdi Ram Lambs
Saudi Journal of Biological Sciences (2014) 21, 133–137
King Saud University
Saudi Journal of Biological Sciences www.ksu.edu.sa www.sciencedirect.com
ORIGINAL ARTICLE
Developmental changes in testicular interstitium in the Najdi Ram Lambs Ahmed A. Al-kawmani a, Mansour M. Alfuraiji a, Faisal M. Abou-Tarboush b, Mohammed A. Alodan a, Mohammad Abul Farah b,* a b
Department of Animal Production, College of Food Sciences and Agriculture, King Saud University, Saudi Arabia Department of Zoology, College of Sciences, King Saud University, Saudi Arabia
Received 16 April 2013; revised 24 August 2013; accepted 1 September 2013 Available online 15 September 2013
KEYWORDS Testicular interstitium; Sexual development; Spermatogenesis; Najdi Lambs
Abstract This experiment was designed to study the relationship between growth rate and sexual development in Najdi ram lambs. Forty-five Najdi ram lambs were used in nine age groups (1–9th month age), keeping 5 lambs in each group. Results show that mean testis size and scrotal circumference increased at a constant rate, reaching a maximum value of 392.00 ± 2.00 g and 25.00 ± 0.32 cm respectively, at the age of 9 months. A similar trend of growth was observed for the mean body weight, reaching a maximum value of 40.2 ± 0.20 kg at the age of 9 months. There was a positive correlation between the increasing rates of body weight and size of the testis. There were significant differences (P < 0.05) in testis size, scrotal circumference and body weight of lambs between all ages (1–9 months). The histological section of testis showed that a small number of mature sperms appeared in the eighth month of age and their numbers increased in the ninth month. In conclusion, the current findings indicated that puberty in Najdi rams occurs between the 8th and 9th months of age. ª 2013 Production and hosting by Elsevier B.V. on behalf of King Saud University.
1. Introduction
* Corresponding author. Address: Department of Zoology, College of Sciences, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia. Tel.: +966 11 4675762; fax: +966 11 4678514. E-mail addresses: [email protected] (A.A. Al-kawmani), [email protected], [email protected] (M.A. Farah). Peer review under responsibility of King Saud University.
Production and hosting by Elsevier
With the increasing interest in the application of artificial insemination in sheep, the semen quality is the main parameter limiting male reproductive efficiency which is influenced by testicular size (Zamiri and Khodaei, 2005). Testis size is a standard of fertility and reproductive ability in the ram, as the amount of testicular mass is correlated with the sperm production ability (Salhab et al., 2001), and also it may be useful as a selection criterion for improving reproductive capacity in both the sexes (Toe et al., 2000; Matos et al., 1992). Furthermore, the rate of testis development in the Najdi ram may be a clear evidence of its sexual performance, and has been correlated
1319-562X ª 2013 Production and hosting by Elsevier B.V. on behalf of King Saud University. http://dx.doi.org/10.1016/j.sjbs.2013.09.001
134 with several reproductive characteristics of female siblings and future female offspring of the ram, such as age at puberty, fertility, prolificacy, and duration of the breeding season (Land and Cart, 1975; Toe et al., 2000). Previous report shows a gradual and linear increase in the testicular volume from 3 to 17 months of age in sheep (Salhab et al., 2001). Scrotal circumference has been described as the best criterion of male sexual development (Notter et al., 1981) but, there is very little evidence correlating age of the ram with scrotal circumference (Notter et al., 1985). In another study, it was reported that various testicular measurements are more correlated with body weight of growing rams than the age (Salhab et al., 2001). A few other reports showed a gradual and rapid increase in both scrotal circumference and testicular volume from 3 to 6 months of age and lambs were in a stage of rapid sexual development as determined by increase in testicular size (Salhab et al., 2001; Ebru, 2005; Ulker et al., 2005). Detailed information about the onset of puberty and sexual maturation are very important and needed for good reproductive management of domestic animals (Ahmad and Noakes, 1996; Bilaspuri and Singh, 1992). Measurements of ram sexual development may be useful for predicting reproductive capacity in rams sires (Katz et al., 1988; Price et al., 2000). Sexual behavior differs between breeds and herd management plays an important role in the speed of the start of puberty (Godfrey et al., 1988; Maina and Katz, 1997; Price et al., 2000, 2001). Although spermatogenesis is an organized process throughout the life of the male (Cheng et al., 2010; Michelle et al., 2009; Leblond and Clermont, 1952), it does not occur simultaneously in all seminiferous tubules but rather in wave-like sequences of maturation, referred to as cycles of the semniferous epithelium. The objectives of this study were to evaluate the testicular development during postnatal period (age from 1–9 months), and to establish an easy and simple technique for assessing the male fertility that could be useful as a tool in breeding programs in Najdi ram lambs. 2. Materials and methods 2.1. Location, animals and diets The experiments were carried out at Al-Watania livestock project near Al-Jouf, 150 km from Domat Al-Jandal, Kingdom of Saudi Arabia (29 480 N, 38 230 E). Forty-five Najdi lambs were used aged 1–9 months (5 rams in each age group). Rams were weaned at approximately 8 weeks of age and maintained on standard growing ration throughout the experiment, with water and mineral blocks available ad libitum. 2.2. Testicular biopsy and histology Prior to biopsy, scrotal circumference was measured by using vernier caliper and testicular volume was estimated using displacement of water from a cup. All rams were weighed to determine the relationship between testis and body weight, and a gonadosomatic index (GSI) was calculated (100 · testis weight/body weight). Then, five rams from each age group were assigned to receive a single testicular biopsy at age 1– 9 months. Testicular biopsy was performed unilaterally on rams under local anesthesia (Lunstra and Echternkamp, 1988).
A.A. Al-kawmani et al. After performing biopsy and taking testicular measurement, the tissues were placed in Bouin’s fixative for 48 h, then immersed in 70% ethanol for another 48 h. After fixation, samples were washed in Dulbecco’s PBS Phosphate Buffered Saline (2 · 1 h), dehydrated through graded percentages of ethanol (50%, 70%, 80%, 95% and 100%; 2 · 1 h each), cleared in xylene (2 · 1 h), pre-infiltrated in a saturated solution of paraffin wax in xylene overnight, infiltrated and were embedded in paraffin wax. Serial sections (5 lm) were made from each sample, mounted onto gelatin-coated glass slides and dried overnight at 37 C. Tissue sections were stained with hematoxylin-eosin and stored at room temperature until the morphometric analysis. The histological preparations were observed using a compound microscope fitted with photographic attachment (Olympus BX41, Japan). All the observations were made at 400· magnification. For the registration of spermatogenesis, the testicular biopsy score count proposed by Johnsen (1970) was followed. 2.3. Statistical analysis Least squares analysis of variance was done using the general linear model (GLM) procedure of the statistical analysis system. Differences between groups were assessed to discriminate among the means (Fisher’s least significant difference: DNKN procedure). In all cases, the level of statistical significance was considered at P < 0.05. 3. Results and discussion This work presents the first description of the age related changes in body growth and sexual development in Najdi ram lambs. The mean of body weight, scrotal circumference, testicular size and relationship between body weight and testicular size are presented in Table 1. These results appear to be in agreement with corresponding data on other breeds of sheep with few differences that appear to be due to effects of the different environment in which rams were raised (Cui et al., 2003). The mean body weight increased at a constant rate, from 1st month of age (7.9 ± 0.46 kg) and reached a maximum value of 40.2 ± 0.20 kg at the age of 9 months. There were significant differences (P < 0.05) between body weights and lambs aged 1–9 months. The relationships between chronological age and body weight are shown in Fig. 1. Obvious differences were observed while comparing the body weight of different sheep breeds at seven months of age. Body weight average score for Najdi rams was 34 kg which is less than the other breeds like Serres (44 kg), Friesland (54 kg), Chios (54 kg) and Karagouniki (50 kg) as reported previously (Belibasaki and Kouimtzis, 2000). Variations in the body weight of different breeds could be attributed to the environment and nutrition. The mean scrotal circumference increased at a constant rate in all lambs, the mean value increased from 1st month of age (6.4 ± 0.24 cm) reaching a maximum value of 25.00 ± 0.32 cm at the age of 9 months. There was a significant difference (P < 0.05) in scrotal circumference values (SC) between all ages (1–7 months) except at 8 and 9 months of ages. The difference in SC was insignificant between these two age groups as shown in
Developmental changes in Najdi Lambs Effect of age on study traits in Najdi ram lambs. (Mean ± SE, n = 5).
Table 1
Body weight (kg)*
Age (month)
Scrotal circumference (cm)*
a
1 2 3 4 5 6 7 8 9 *
135
7.9 ± 0.46 15.0 ± 0.32b 18.2 ± 0.58c 23.2 ± 0.58d 25.6 ± 0.24e 31.2 ± 0.24f 34.2 ± 0.37g 37.4 ± 0.24h 40.2 ± 0.20i
6.40 ± 0.24 9.40 ± 0.51b 12.00 ± 0.54c 13.60 ± 0.24d 16.20 ± 0.37e 20.60 ± 0.24f 22.40 ± 0.24g 24.40 ± 0.24h 25.00 ± 0.32h
y = 3.94x + 6.1778 R² = 0.9875
45 40 Body Weight (kg)
GSI*
a
0.56 ± 0.007a 0.44 ± 0.032b 0.52 ± 0.017a 0.44 ± 0.014b 0.92 ± 0.001c 0.96 ± 0.006d 0.97 ± 0.001d 0.97 ± 0.001d 0.98 ± 0.001d
44.00 ± 2.45 66.00 ± 5.09b 95.00 ± 2.64c 103.00 ± 3.64c 236.00 ± 2.45d 302.00 ± 2.00e 332.00 ± 3.74f 364.00 ± 2.44g 392.00 ± 2.00h
Different superscripts (a-i) in the same column are significantly different (P < 0.05) GSI = gonadosomatic index.
50
35 30 25 20 15 10 5 0
0
1
2
3
4
5
6
7
8
9
Age in Months
Figure 1 Differences in mean body weight in Najdi ram lambs aged 1–9 months (n = 5).
Table 1. The relationship between chronological age and scrotal circumference is shown in Fig. 2. Comparison of scrotal circumference in different sheep breeds at the age of seven months also revealed a clear difference. Average score for scrotal circumference observed in Najdi breeds was 22.40 cm which was less than the value observed in other breeds like Serres (25 cm), Friesland (30 cm), Chios (27 cm) and Karagouniki (25 cm) as has been reported earlier by Belibasaki and Kouimtzis (2000). Lower scrotal circumference score for Najdi
rams also correlated well with its body weight compared to the other breeds. As suggested before, these variations could have been the effect of the environment where the lambs were being raised and nutrition which was provided. In all Najdi lambs, the mean testis size increased at a constant rate from the 1st month of age (44.0 ± 2.45 g) to the 9th month (392.00 ± 2.00 g). There was a significant difference (P < 0.05) in the testis size (TS) of Najdi rams between the ages of 1, 2, 5, 6, 7, 8 and 9 months, except at the age of 3 and 4 months where the TS was not significantly different between these two age groups, as shown in Table 1. The testicular size increased rapidly between 4 and 6 months, followed by a period of gradual growth. The relationship between the chronological age and testis size is shown in Fig. 3. There was a positive correlation between the body weight and the testicular size gonadosomatic index (GSI) in Najdi rams as shown in Table 1. Increase in the body weight always leads to an increase in the testicular size. The differences were significant between ages 1, 2, 5, and 6 months (P < 0.05), while there were no significant differences between ages 6, 7, 8, and 9 months, between ages 1 and 3 months and between ages 2 and 4 months. Spermatogenesis was assessed using Johnsen-like score (Johnsen, 1970). The results show that each age-group has a different histological structure showing the presence of spermatozoa, spermatids, spermatocytes, spermatogonia and Sertoli cells, except for the second and third month-age groups which did not show any difference in the structure.
500
30 25 20.6
22.4
24.4
20 16.2 15 10
12
450
25
400
Tess Weight (g)
Scrotal Circumference (cm)
Testis weight (g)*
a
13.6
9.4 6.4
350 300 250 200 150 100
5
50
0
1
2
3
4
5 6 Age in Months
7
8
9
Figure 2 The relationships between scrotal circumference values and different ages (1–9 months) in Najdi ram lambs (n = 5).
0 1
2
3
4
5
6
7
8
9
Age in Months
Figure 3 The relationship between testis weight at different ages (1–9 months) in Najdi ram lambs (n = 5).
136
A.A. Al-kawmani et al.
Figure 4 Cross section of testis showing the process of spermatogenesis in the Najdi ram lamb at different age (2–9 months), (A) two months of age, showing immature spermatogonia and sertoli cells, (Johnsen-like score 3), (B) three months of age, showing spermatogonia, (Johnsen-like score 3), (C) four months of age, showing spermatogonia, (Johnsen-like score 3), (D) five months of age, few spermatocytes were observed (Johnsen-like score 4). (E) six months of age, more spermatocytes were present (Johnsen-like score 5), (F) seven months of age, showing spermatids (Johnsen-like score 6,7), (G) eight months of age, showing few spermatozoa (Johnsen-like score 8,9), (H) nine months of age, showing uniformly distributed Spermatozoa (Johnsen-like score 10). Abbreviations: (St) seminiferous tubules, (Sg) spermatogonia, (Sc) spermatocytes, (Rs) spermatids, (Sp) spermatozoa, (Le) leydig cells, (Se) sertoli cells. Magnification 400·.
Developmental changes in Najdi Lambs During the 2nd, 3rd, and fourth months of age there were spermatogonia and Sertoli cells, but there was no cell division within the seminiferous tubules except for an increase in the seminiferous tubule diameter at the expense of cavity ledge cells with reduced numbers of seminiferous tubules in sections as shown in Fig. 4. In the fifth month of age, only a few spermatocytes (<5) and no spermatids or spermatozoa (Spermatozoa are defined as cells having achieved the small head form of spermatozoon) were present. In the sixth month of age no spermatozoa or spermatids but several sprermatocytes were found. In the seventh month of age no spermatozoa but many spermatids were present. In the eighth month of age many spermatozoa were present but germinal epithelium was disorganized with marked sloughing or obliteration of lumen. In the ninth month of age complete spermatogenesis was observed with the presence of many spermatozoa. Germinal epithelium was organized in a regular thickness leaving an open lumen as shown in Fig. 4. The results showed that Najdi rams reach puberty between eight and nine months of age similar to the Tanyang breed (Cui et al., 2003), whereas Chios, Karagouniki, Serres and Friesland breed rams reach puberty between six and seven months of age (Belibasaki and Kouimtzis, 2000). Late onset of puberty in Najdi breeds compared to other breeds could be attributed to poor nutrition and/or poor care in the breeding farm where they were raised. Another possibility is that late puberty is intrinsic to Najdi breed which is a subject of further study. Acknowledgements This work was funded by a grant from the Deanship of Scientific Research at King Saud University, which is gratefully acknowledged. References Ahmad, N., Noakes, D.E., 1996. Sexual maturity in British breeds of goat kids. Br. Vet. J. 5, 93–103. Belibasaki, S., Kouimtzis, S., 2000. Sexual activity and body and testis growth in prepubertal ram lambs of Friesland, Chios, Karagouniki and Serres dairy sheep in Greece. Small Rumin. Res. 37, 109–113. Bilaspuri, G.S., Singh, K., 1992. Developmental changes in body weight and testicular characteristics in Malabari goat kids. Theriogenology 37, 507–520. Cheng, C.Y., Wong, E.W., Helen, H.N., Yan, H.H., Mruk, D.D., 2010. Regulation of spermatogenesis in the microenvironment of the seminiferous epithelium: new insights and advances. Mol. Cell. Endocrinol. 315, 49–56. Cui, S., Chen, Y.F., Yue, H.N., He, Y.Q., McNeilly, A.S., 2003. Sexual development and the effects of active immunization against GnRH in Chinese Tanyang ram lambs. Anim. Reprod. Sci. 77, 129–139. Ebru, E., 2005. Testicular development and body weight gain from birth to 1 year of age of Awassi and Redkaraman sheep and their reciprocal crosses. Small Rumin. Res. 59, 79–82.
137 Godfrey, R.W., Collins, J.R., Gray, M.L., 1988. Evaluation of sexual behaviour of hair sheep rams in a tropical environment. J. Anim. Sci. 76, 714–717. Johnsen, S.G., 1970. Testicular biopsy score count-a method for registration of spermatogenesis in human testes: normal values and results in 335 hypogonadal males. Hormones 1, 2–25. Katz, L.S., Price, E.O., Wallach, S.J., Zenchak, J.J., 1988. The relationship of male-male mounting to the sexual preferences of young rams. J. Anim. Sci. 66, 1166–1173. Land, R.B., Cart, W.R., 1975. Testis growth and plasma LH concentration following hemicastration and its relation with female prolificacy in sheep. J. Reprod. Fertil. 45, 495–501. Leblond, C.P., Clermont, Y., 1952. Definition of the stages of the cycle of the seminiferous epithelium in the rat. Ann. N. Y. Acad. Sci. 55, 548–573. Lunstra, D.D., Echternkamp, S.E., 1988. Repetitive testicular biopsy in the ram during pubertal development. Theriogenology 29, 803– 810. Maina, D., Katz, L.S., 1997. Exposure to a recently mated male increases ram sexual performance. Appl. Anim. Behav. Sci. 51, 69– 74. Matos, C.A., Thomas, D.L., Nash, T.G., Waldron, D.F., Stookey, J.M., 1992. Genetic analyses of scrotal circumference size and growth in Rambouillet lambs. J. Anim. Sci. 70, 43– 50. Michelle, W.M., Dolores, D.M., Will, M.L., Cheng, C.Y., 2009. Cytokines and junction restructuring events during spermatogenesis in the testis: an emerging concept of regulation. Cytokine Growth Factor Rev. 20, 329–338. Notter, D.R., Lucas, J.R., McClaugherg, F.S., 1981. Accuracy of estimation of testis weight from in situ testis measures in ram lambs. Theriogenology 15, 227–234. Notter, D.R., Lucas, J.R., McClaugherty, F.S., Copenhaver, J.S., 1985. Breed group differences in testicular growth patterns in spring-born ram lambs. J. Anim. Sci. 60, 622–631. Price, E.O., Bench, C.J., Borgwardt, R.E., Dally, M.R., 2000. Sexual performance of twin ram lambs and the effect of number and sex of contemporary siblings. Appl. Anim. Behav. Sci. 68, 199–205. Price, E.O., Borgwardt, R.E., Dally, M.R., 2001. Male-male competition fails to sexually stimulate domestic rams. Appl. Anim. Behav. Sci. 74, 217–222. Salhab, S.A., Zarkawi, M., Wardeh, M.F., Al-Masri, M.R., Kassem, R., 2001. Development of testicular dimensions and size, and their relationship to age, body weight an parental size in growing Awassi ram lambs. Small Rumin. Res. 40, 187–191. Toe, F., Rege, J.E., Mukasa-Mugerwa, E., Tembely, S., Anindo, D., Baker, R.L., Lahlou-Kassi, A., 2000. Reproductive characteristics of Ethiopian highland sheep I. Genetic parameters of testicular measurements in ram lambs and relationship with age at puberty in ewe lambs. Small Rumin. Res. 36, 227–240. Ulker, H., Kanter, M., Go¨kdal, O., Aygu¨n, T., Karakusß, F., Sakarya, M.E., deAvila, D.M., Reeves, J.J., 2005. Testicular development, ultrasonographic and histological appearance of the testis in ram lambs immunized against recombinant LHRH fusion proteins. Anim. Reprod. Sci. 86, 205–219. Zamiri, M.J., Khodaei, H.R., 2005. Seasonal thyroidal activity and reproductive characteristic of Iranian fat-tailed rams. Anim. Reprod. Sci. 88, 245–255.
King Saud University
Saudi Journal of Biological Sciences www.ksu.edu.sa www.sciencedirect.com
ORIGINAL ARTICLE
Developmental changes in testicular interstitium in the Najdi Ram Lambs Ahmed A. Al-kawmani a, Mansour M. Alfuraiji a, Faisal M. Abou-Tarboush b, Mohammed A. Alodan a, Mohammad Abul Farah b,* a b
Department of Animal Production, College of Food Sciences and Agriculture, King Saud University, Saudi Arabia Department of Zoology, College of Sciences, King Saud University, Saudi Arabia
Received 16 April 2013; revised 24 August 2013; accepted 1 September 2013 Available online 15 September 2013
KEYWORDS Testicular interstitium; Sexual development; Spermatogenesis; Najdi Lambs
Abstract This experiment was designed to study the relationship between growth rate and sexual development in Najdi ram lambs. Forty-five Najdi ram lambs were used in nine age groups (1–9th month age), keeping 5 lambs in each group. Results show that mean testis size and scrotal circumference increased at a constant rate, reaching a maximum value of 392.00 ± 2.00 g and 25.00 ± 0.32 cm respectively, at the age of 9 months. A similar trend of growth was observed for the mean body weight, reaching a maximum value of 40.2 ± 0.20 kg at the age of 9 months. There was a positive correlation between the increasing rates of body weight and size of the testis. There were significant differences (P < 0.05) in testis size, scrotal circumference and body weight of lambs between all ages (1–9 months). The histological section of testis showed that a small number of mature sperms appeared in the eighth month of age and their numbers increased in the ninth month. In conclusion, the current findings indicated that puberty in Najdi rams occurs between the 8th and 9th months of age. ª 2013 Production and hosting by Elsevier B.V. on behalf of King Saud University.
1. Introduction
* Corresponding author. Address: Department of Zoology, College of Sciences, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia. Tel.: +966 11 4675762; fax: +966 11 4678514. E-mail addresses: [email protected] (A.A. Al-kawmani), [email protected], [email protected] (M.A. Farah). Peer review under responsibility of King Saud University.
Production and hosting by Elsevier
With the increasing interest in the application of artificial insemination in sheep, the semen quality is the main parameter limiting male reproductive efficiency which is influenced by testicular size (Zamiri and Khodaei, 2005). Testis size is a standard of fertility and reproductive ability in the ram, as the amount of testicular mass is correlated with the sperm production ability (Salhab et al., 2001), and also it may be useful as a selection criterion for improving reproductive capacity in both the sexes (Toe et al., 2000; Matos et al., 1992). Furthermore, the rate of testis development in the Najdi ram may be a clear evidence of its sexual performance, and has been correlated
1319-562X ª 2013 Production and hosting by Elsevier B.V. on behalf of King Saud University. http://dx.doi.org/10.1016/j.sjbs.2013.09.001
134 with several reproductive characteristics of female siblings and future female offspring of the ram, such as age at puberty, fertility, prolificacy, and duration of the breeding season (Land and Cart, 1975; Toe et al., 2000). Previous report shows a gradual and linear increase in the testicular volume from 3 to 17 months of age in sheep (Salhab et al., 2001). Scrotal circumference has been described as the best criterion of male sexual development (Notter et al., 1981) but, there is very little evidence correlating age of the ram with scrotal circumference (Notter et al., 1985). In another study, it was reported that various testicular measurements are more correlated with body weight of growing rams than the age (Salhab et al., 2001). A few other reports showed a gradual and rapid increase in both scrotal circumference and testicular volume from 3 to 6 months of age and lambs were in a stage of rapid sexual development as determined by increase in testicular size (Salhab et al., 2001; Ebru, 2005; Ulker et al., 2005). Detailed information about the onset of puberty and sexual maturation are very important and needed for good reproductive management of domestic animals (Ahmad and Noakes, 1996; Bilaspuri and Singh, 1992). Measurements of ram sexual development may be useful for predicting reproductive capacity in rams sires (Katz et al., 1988; Price et al., 2000). Sexual behavior differs between breeds and herd management plays an important role in the speed of the start of puberty (Godfrey et al., 1988; Maina and Katz, 1997; Price et al., 2000, 2001). Although spermatogenesis is an organized process throughout the life of the male (Cheng et al., 2010; Michelle et al., 2009; Leblond and Clermont, 1952), it does not occur simultaneously in all seminiferous tubules but rather in wave-like sequences of maturation, referred to as cycles of the semniferous epithelium. The objectives of this study were to evaluate the testicular development during postnatal period (age from 1–9 months), and to establish an easy and simple technique for assessing the male fertility that could be useful as a tool in breeding programs in Najdi ram lambs. 2. Materials and methods 2.1. Location, animals and diets The experiments were carried out at Al-Watania livestock project near Al-Jouf, 150 km from Domat Al-Jandal, Kingdom of Saudi Arabia (29 480 N, 38 230 E). Forty-five Najdi lambs were used aged 1–9 months (5 rams in each age group). Rams were weaned at approximately 8 weeks of age and maintained on standard growing ration throughout the experiment, with water and mineral blocks available ad libitum. 2.2. Testicular biopsy and histology Prior to biopsy, scrotal circumference was measured by using vernier caliper and testicular volume was estimated using displacement of water from a cup. All rams were weighed to determine the relationship between testis and body weight, and a gonadosomatic index (GSI) was calculated (100 · testis weight/body weight). Then, five rams from each age group were assigned to receive a single testicular biopsy at age 1– 9 months. Testicular biopsy was performed unilaterally on rams under local anesthesia (Lunstra and Echternkamp, 1988).
A.A. Al-kawmani et al. After performing biopsy and taking testicular measurement, the tissues were placed in Bouin’s fixative for 48 h, then immersed in 70% ethanol for another 48 h. After fixation, samples were washed in Dulbecco’s PBS Phosphate Buffered Saline (2 · 1 h), dehydrated through graded percentages of ethanol (50%, 70%, 80%, 95% and 100%; 2 · 1 h each), cleared in xylene (2 · 1 h), pre-infiltrated in a saturated solution of paraffin wax in xylene overnight, infiltrated and were embedded in paraffin wax. Serial sections (5 lm) were made from each sample, mounted onto gelatin-coated glass slides and dried overnight at 37 C. Tissue sections were stained with hematoxylin-eosin and stored at room temperature until the morphometric analysis. The histological preparations were observed using a compound microscope fitted with photographic attachment (Olympus BX41, Japan). All the observations were made at 400· magnification. For the registration of spermatogenesis, the testicular biopsy score count proposed by Johnsen (1970) was followed. 2.3. Statistical analysis Least squares analysis of variance was done using the general linear model (GLM) procedure of the statistical analysis system. Differences between groups were assessed to discriminate among the means (Fisher’s least significant difference: DNKN procedure). In all cases, the level of statistical significance was considered at P < 0.05. 3. Results and discussion This work presents the first description of the age related changes in body growth and sexual development in Najdi ram lambs. The mean of body weight, scrotal circumference, testicular size and relationship between body weight and testicular size are presented in Table 1. These results appear to be in agreement with corresponding data on other breeds of sheep with few differences that appear to be due to effects of the different environment in which rams were raised (Cui et al., 2003). The mean body weight increased at a constant rate, from 1st month of age (7.9 ± 0.46 kg) and reached a maximum value of 40.2 ± 0.20 kg at the age of 9 months. There were significant differences (P < 0.05) between body weights and lambs aged 1–9 months. The relationships between chronological age and body weight are shown in Fig. 1. Obvious differences were observed while comparing the body weight of different sheep breeds at seven months of age. Body weight average score for Najdi rams was 34 kg which is less than the other breeds like Serres (44 kg), Friesland (54 kg), Chios (54 kg) and Karagouniki (50 kg) as reported previously (Belibasaki and Kouimtzis, 2000). Variations in the body weight of different breeds could be attributed to the environment and nutrition. The mean scrotal circumference increased at a constant rate in all lambs, the mean value increased from 1st month of age (6.4 ± 0.24 cm) reaching a maximum value of 25.00 ± 0.32 cm at the age of 9 months. There was a significant difference (P < 0.05) in scrotal circumference values (SC) between all ages (1–7 months) except at 8 and 9 months of ages. The difference in SC was insignificant between these two age groups as shown in
Developmental changes in Najdi Lambs Effect of age on study traits in Najdi ram lambs. (Mean ± SE, n = 5).
Table 1
Body weight (kg)*
Age (month)
Scrotal circumference (cm)*
a
1 2 3 4 5 6 7 8 9 *
135
7.9 ± 0.46 15.0 ± 0.32b 18.2 ± 0.58c 23.2 ± 0.58d 25.6 ± 0.24e 31.2 ± 0.24f 34.2 ± 0.37g 37.4 ± 0.24h 40.2 ± 0.20i
6.40 ± 0.24 9.40 ± 0.51b 12.00 ± 0.54c 13.60 ± 0.24d 16.20 ± 0.37e 20.60 ± 0.24f 22.40 ± 0.24g 24.40 ± 0.24h 25.00 ± 0.32h
y = 3.94x + 6.1778 R² = 0.9875
45 40 Body Weight (kg)
GSI*
a
0.56 ± 0.007a 0.44 ± 0.032b 0.52 ± 0.017a 0.44 ± 0.014b 0.92 ± 0.001c 0.96 ± 0.006d 0.97 ± 0.001d 0.97 ± 0.001d 0.98 ± 0.001d
44.00 ± 2.45 66.00 ± 5.09b 95.00 ± 2.64c 103.00 ± 3.64c 236.00 ± 2.45d 302.00 ± 2.00e 332.00 ± 3.74f 364.00 ± 2.44g 392.00 ± 2.00h
Different superscripts (a-i) in the same column are significantly different (P < 0.05) GSI = gonadosomatic index.
50
35 30 25 20 15 10 5 0
0
1
2
3
4
5
6
7
8
9
Age in Months
Figure 1 Differences in mean body weight in Najdi ram lambs aged 1–9 months (n = 5).
Table 1. The relationship between chronological age and scrotal circumference is shown in Fig. 2. Comparison of scrotal circumference in different sheep breeds at the age of seven months also revealed a clear difference. Average score for scrotal circumference observed in Najdi breeds was 22.40 cm which was less than the value observed in other breeds like Serres (25 cm), Friesland (30 cm), Chios (27 cm) and Karagouniki (25 cm) as has been reported earlier by Belibasaki and Kouimtzis (2000). Lower scrotal circumference score for Najdi
rams also correlated well with its body weight compared to the other breeds. As suggested before, these variations could have been the effect of the environment where the lambs were being raised and nutrition which was provided. In all Najdi lambs, the mean testis size increased at a constant rate from the 1st month of age (44.0 ± 2.45 g) to the 9th month (392.00 ± 2.00 g). There was a significant difference (P < 0.05) in the testis size (TS) of Najdi rams between the ages of 1, 2, 5, 6, 7, 8 and 9 months, except at the age of 3 and 4 months where the TS was not significantly different between these two age groups, as shown in Table 1. The testicular size increased rapidly between 4 and 6 months, followed by a period of gradual growth. The relationship between the chronological age and testis size is shown in Fig. 3. There was a positive correlation between the body weight and the testicular size gonadosomatic index (GSI) in Najdi rams as shown in Table 1. Increase in the body weight always leads to an increase in the testicular size. The differences were significant between ages 1, 2, 5, and 6 months (P < 0.05), while there were no significant differences between ages 6, 7, 8, and 9 months, between ages 1 and 3 months and between ages 2 and 4 months. Spermatogenesis was assessed using Johnsen-like score (Johnsen, 1970). The results show that each age-group has a different histological structure showing the presence of spermatozoa, spermatids, spermatocytes, spermatogonia and Sertoli cells, except for the second and third month-age groups which did not show any difference in the structure.
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Figure 2 The relationships between scrotal circumference values and different ages (1–9 months) in Najdi ram lambs (n = 5).
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Figure 3 The relationship between testis weight at different ages (1–9 months) in Najdi ram lambs (n = 5).
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A.A. Al-kawmani et al.
Figure 4 Cross section of testis showing the process of spermatogenesis in the Najdi ram lamb at different age (2–9 months), (A) two months of age, showing immature spermatogonia and sertoli cells, (Johnsen-like score 3), (B) three months of age, showing spermatogonia, (Johnsen-like score 3), (C) four months of age, showing spermatogonia, (Johnsen-like score 3), (D) five months of age, few spermatocytes were observed (Johnsen-like score 4). (E) six months of age, more spermatocytes were present (Johnsen-like score 5), (F) seven months of age, showing spermatids (Johnsen-like score 6,7), (G) eight months of age, showing few spermatozoa (Johnsen-like score 8,9), (H) nine months of age, showing uniformly distributed Spermatozoa (Johnsen-like score 10). Abbreviations: (St) seminiferous tubules, (Sg) spermatogonia, (Sc) spermatocytes, (Rs) spermatids, (Sp) spermatozoa, (Le) leydig cells, (Se) sertoli cells. Magnification 400·.
Developmental changes in Najdi Lambs During the 2nd, 3rd, and fourth months of age there were spermatogonia and Sertoli cells, but there was no cell division within the seminiferous tubules except for an increase in the seminiferous tubule diameter at the expense of cavity ledge cells with reduced numbers of seminiferous tubules in sections as shown in Fig. 4. In the fifth month of age, only a few spermatocytes (<5) and no spermatids or spermatozoa (Spermatozoa are defined as cells having achieved the small head form of spermatozoon) were present. In the sixth month of age no spermatozoa or spermatids but several sprermatocytes were found. In the seventh month of age no spermatozoa but many spermatids were present. In the eighth month of age many spermatozoa were present but germinal epithelium was disorganized with marked sloughing or obliteration of lumen. In the ninth month of age complete spermatogenesis was observed with the presence of many spermatozoa. Germinal epithelium was organized in a regular thickness leaving an open lumen as shown in Fig. 4. The results showed that Najdi rams reach puberty between eight and nine months of age similar to the Tanyang breed (Cui et al., 2003), whereas Chios, Karagouniki, Serres and Friesland breed rams reach puberty between six and seven months of age (Belibasaki and Kouimtzis, 2000). Late onset of puberty in Najdi breeds compared to other breeds could be attributed to poor nutrition and/or poor care in the breeding farm where they were raised. Another possibility is that late puberty is intrinsic to Najdi breed which is a subject of further study. Acknowledgements This work was funded by a grant from the Deanship of Scientific Research at King Saud University, which is gratefully acknowledged. References Ahmad, N., Noakes, D.E., 1996. Sexual maturity in British breeds of goat kids. Br. Vet. J. 5, 93–103. Belibasaki, S., Kouimtzis, S., 2000. Sexual activity and body and testis growth in prepubertal ram lambs of Friesland, Chios, Karagouniki and Serres dairy sheep in Greece. Small Rumin. Res. 37, 109–113. Bilaspuri, G.S., Singh, K., 1992. Developmental changes in body weight and testicular characteristics in Malabari goat kids. Theriogenology 37, 507–520. Cheng, C.Y., Wong, E.W., Helen, H.N., Yan, H.H., Mruk, D.D., 2010. Regulation of spermatogenesis in the microenvironment of the seminiferous epithelium: new insights and advances. Mol. Cell. Endocrinol. 315, 49–56. Cui, S., Chen, Y.F., Yue, H.N., He, Y.Q., McNeilly, A.S., 2003. Sexual development and the effects of active immunization against GnRH in Chinese Tanyang ram lambs. Anim. Reprod. Sci. 77, 129–139. Ebru, E., 2005. Testicular development and body weight gain from birth to 1 year of age of Awassi and Redkaraman sheep and their reciprocal crosses. Small Rumin. Res. 59, 79–82.
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