Analysis of Testicular Histological Structure in Lambs
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Testicular Histological Structure and Scrotal During Postnatal Development till puberty Circumference in Najdi and Naemi Ram Lambs
- Ahmed A. Al-kawmania,,. Faisal M. Abou-Tarbousha,. Mohammad Abul Faraha,. Mansour M. Alfuraijib,. Salah A. Ibrahima.
The present work is conducted to elucidate the relationship among chronological age, testis size, and the postnatal development of the seminiferous epithelium of the testes of the Najdi and Naemi ram lams from 1 month to 9 months of age. A total of 90 (45 Najdi and 45 Naimi ram lambs) were used aged 1, 2, 3, 4, 5, 6, 7, 8 and 9 months (5 lambs in each age from both breeds ). The results showed that testicular size increased rapidly between 3 and 6 months, followed by a period of gradual growth. There was a significant (P < 0.05) difference in testis size (TS) in ram lambs between different age groups, similarly there were significant differences between body weights and lambs age at 1 to 9 months. There was a positive correlation between the increasing rates of body weight and size of the testicle. Microscopically, each age-group has a different histological appearance except at the 2, 3 and 4 in Najdi and 2,3,4 and 5 in Naemi month age-groups which showed few differences. Small quantities of spermatozoa appeared in the eighth month of age and increased in the ninth month. In conclusion, the current findings indicate that puberty in Najdi and Naemi rams occurs at the 8th and 9th months of age, respectively.
- Testicular histology;. Najdi Lambs;. Naemi lambs;. Testis size.
Testis size may be useful as a selection criterion for improving reproductive capacity in both sheep sexes, (Toe et al., 2000; Matos et al., 1992); however, detailed information about the onset of puberty and sexual maturity are needed for good reproductive management of domestic animals (Nishimura et al., 2000). Measurements of ram sexual development parameters may be useful for predicting reproductive capacity in rams (Kridli and Said, 1999; Price et al.,2000). Characterization of puberty and early sexual development are important criteria used in the selection of ram-lambs within a breed. (Kridli and Al-Yacoub, 2006). Most breeds of sheep are seasonal breeders, but the age of puberty in males differs from one breed to another (Foster and Ryan, 1990; Foster et al., 2002). The ram age of 4–5 months of age is a critical period for pubertal changes and testosterone plays an important role in the appearance of sexual desire of ram lambs ( Cui et al., 2003 ) as determined by the first existence of the spermatozoa in ejaculated semen. Postnatal development and function of the testes are controlled by a complex interaction of circulating gonadotropins with cytokines and growth factors (Bilgin et al., 2004). and establishment of hypothalamo-hypophyseal gonadal axis. Therefore, this work was designed to determine testicular changes occurred during the postnatal period until puberty is attained.
Materials and Methods
The experiments were carried out at Al-Watania Livestock in Bosaitta area Project near to Al-Jouf, Kingdom of Saudi Arabia (29°.00 – 48.00’N, 38°.00 – 23.00’ E). A total 0f 90 (45 Najdi and 45 Naimi ram lambs) were used aged 1, 2, 3, 4, 5, 6, 7, 8 and 9 months (5 rams in each age group from both breeds ). Rams were weaned at approximately 8 weeks of age and maintained on standard growing ration throughout the experiment, water and mineral block were available ad libitum. Testicular samples were collected in March i.e. 90 a total samples for all animal in all age groups. (original 5 samples per each age group). Testicular biopsies were collected from each ram lambs of both breeds by operative removal of a piece of tissue from the testis, so small as to have no deleterious effect on the testis yet large enough to include a representative group of tubules.
Prior to biopsy, scrotal circumference was measured by using vernier caliper and testicular size was estimated by displacement of water. Body weight (BW) of each ram lamb was recorded at the commencement of the experiment and once a time every month. Then, one ram lambs from each age group in both breeds was assigned to obtain a single testicular biopsy was at ages 1, 2, 3, 4, 5, 6, 7, 8, and 9 months. Testicular biopsy performed unilaterally on rams under local anesthesia (Lunstra and Echternkamp, 1988). The site of the puncture was corresponded to the middle of the testis, on the side opposite to the epididymis, an avascular area of the tunica vaginalis. A specific 14-G needle was introduced lightly and carefully against the testis.
Upon completion of testicles measurements and biopsy collection samples were prepared for histological procedure by fixing them in bouns fixative for 48 hours followed by 70% alcohol and Dulbecco’s Phosphate Buffered Saline (PBS) (2 × 1 h) washing, respectively. Samples were then processed overnight for dehydration with increasing concentration of alcohols series, clearing with three changes of xylene and impregnated with melted paraffin wax using automatic tissue processor (VIP tissue, Tek 5 Jr, Sakura, Japan) .The processed tissue samples were embedded in paraffin blocks and four micron thickness serial sections were obtained with Rotary microtome (RM2245, Leica, Germany). The sections were stained with routine hematoxylin and eosin stains (H&E) and mounted with DPX by using Autostainer machine (Leica ST 5020, Lecia Company, Germany) and stored at room temperature till quantitative morphometric analysis and microscopic examination and photogray were done, (DelVento et al., 1992).
Least square analysis of variance was done using the general linear model (GLM) procedure of the statistical analysis system. Differences among groups were assessed to check significance among the means (Fisher's least significant difference: (LSD) and Duncan, 1955 procedure). In all cases, the level of statistical significance was considered at P < 0.05.
The statistical analysis revealed significant (P < 0.05) differences for breed (B) effect as well as age (A) effect and B × A interaction on testicular measurements and body weight of 1 to 9 month-old (Table,1.2).
The mean body weight in Najdi and Naemi increased at a constant rate, reaching a maximum value of 40.2±0.20 kg and 32.80±0.20 kg at the age of 9 months, respectively. There were significant differences (P < 0.05) in body weight with the advance-ement of age in both breeds. The relationship between chronological age and body weight is shown in Fig. (1). The Najdi body weight mean was greater than that of Naemi from first to ninth month with significant differences (Table,1.2).
The mean scrotal circumference in Najdi and Naemi increased at a constant rate, reaching a maximum value of 25.00±0.32 cm and 22.6±0.24cm at the age of 9 months, respectively. There were significant differences (P< 0.05) in scrotal circumference with lambs age of both breeds at 1, 2, 3, 4, 5, 6, 7, 8 and 9 months. The relationship among chronological age and scrotal circumference is shown in Fig. (2). The Najdi mean scrotal circumference was greater than that of Naemi from first to seventh month with significant differences (P < 0.05).
The mean testis size in Najdi and Naemi increased at a constant rate, reaching a maximum value of 392.00±2.00 g and 316.00±2.45g at the age of 9 months respectively. There were significant differences (P < 0.05) between testis size and lambs age of both breeds at 1, 2, 3, 4, 5, 6, 7, 8 and 9 months. The relationship between chronological age and testis size is shown in Fig.3. The Najdi mean testis size was greater than size of Naemi from first to ninth month with significant differences were presented as shown in Table,1.2 (P< 0.05).
In Najdi histological sections showed no cellular divisions for spermatogonia within the seminiferous tubules during the second, third,and fourth months of age except an increase in the seminiferous tubules diameter at the expense of cavity with the advancement of age. Interstitial tissue containing Leydig cells was clearly observed. In Naemi ram lambs, the histological sections showed no divisions for spermatogonia inside the seminiferous tubules during the second, third, fourth and fifth months of age, with a small expansion in the seminiferous tubules diameter and there was Sertoli cells and Leydig cells were increasing observed as shown in Fig. 4.
At the fifth month of age najdi ram lambs there were few large diameter spermatocytes in the sections as a result of the growing phase of spermatocytogenesis. At the sixth month of age, no spermatozoa but several sprermatocytes and few spermatids were existed as a result of initiation of meiotic division as shown in Fig. 4. At the seventh month of age no spermatozoa but all types of spermatogenic cells, especially spermatids could be seen
While in the Naemi ram lambs the process of cellular division of spermatogonia was delayed to the sixth month of age spermatocytes, spermatids or spermatozoa were no present as shown in Fig. 4. At the seventh month of age no spermatozoa but few spermatocytes were observed. At the eighth month of age no spermatozoa but many spermatids were present as shown in Fig. 4. At the ninth month of age some spermatozoa were seen within the lumen of seminiferous tubules but germinal epithelium was disorganized with marked sloughing or obliteration of lumen as shown in Fig. 4.
While Najdi ramp-lambs at the eighth month of age many spermatozoa were present but germinal epithelium disorganized with marked sloughing or obliteration of lumen as shown in Fig. 4. At the ninth month of age, complete spermatogenesis with many spermatozoa were clearly observed. Germinal epithelium was organized in a regular thickness leaving a relatively small open lumen as shown in Fig.4.
The results of the present work showed the existence of significant monthly-based differences in body weight (BW) of Najdi and Naemi ram lambs from 1stto 9th months of age where BW in Najdi was greater than that of Naemi in all a singed months. These results are in agreement with corresponding data on other studies (Cui et al, 2003), with few differences that appear to be due to effects of breed and environment in which rams were raised. In the research, there was an increase in body weight at a constant rate in both breed as the animal progresses in age until reaches sexual maturity and fully developed body (Emsen, 2005; Belibasaki and Kouimtzis, 2000). However, the increase rate of body weight is mainly dependent on the sheep breeds.
A gradual and rapid increase in scrotal circumference in both Najdi and Naemi ram lambs was observed from 3 to 6 months of age as determined by increase in testicular size. These results are similar to those obtained by several researchers. Emsen (2005) found an increase in both scrotal circumference and testicular volume from 3 to 6 month of age in Awassi and Redkaraman sheep. In an others, study (Salhab et al., 2002) in Awassi ram lambs reported a sharp increase in testicular dimensions started around 7 month of age and at average body weight of 34.6 kg. From all these data, it could be suggested that from 3 to 6 months of age, lambs were in a stage of rapid sexual development. The scrotal circumference (SC) in Najdi and Naemi breeds was 25cm and 21cm, respectively while in other breeds at the same age it was 25cm Serres, 30cm Friesland , 27cm Chios and 25cm Karagouniki (Belibasaki and Kouimtzis, 2000). These differences may be due to environmental differences, nutritional and breed effects. In the previous four breeds studied there was a significant breed difference in SC. At puberty the SC was 33.9 cm in Friesland lambs, 4- 6 cm greater than in the three breeds. Friesland ram lambs had a greater SC throughout the entire period studied.
In all Najdi ram lambs, a gradual and rapid increase was observed in the mean testis size from 3 to 7 months of age, while in Naemi ram lambs it occurred from 4 to 7 months of age. These data suggest that from 3 to 7 months of age, lambs were in a stage of rapid sexual development as determined by an increase in testicular size. These results are similar to those obtained by Emsen, (2005) and Ulker et al., (2005).
The increase in testicular size, length, width and circumference between 3 and 7 months of age was in accordance with the development of body weight, while the increase of testicular volume was three times greater than the growth of body weight or the three other testicular parameters in a study conducted by Salhab et al. (2002).
In Najdi and Naemi ram lambs there was a positive relationship between body weight and testicular size. Salhab et al. (2002) found that testes measurements were progressively correlated with body weight more than with age. There were a clear differences in testis size for different sheep breeds at different months of age (Belibasaki and Kouimtzis, 2000; Koyuncu et al., 2005; Emsen, 2005 ). Thus, the early pubertal development associated with increased body weight due to crossbreeding is desirable in terms of improved reproductive performance.
In Najdi ram lambs as shown in Fig.(4), there was no clear difference in spermatogonia during the 2nd, 3rd, and 4th months of age while in Naemi ram lambs, there was no clear difference in spermatogonia during the 2nd, 3rd, 4th and 5th months of age and there was no any kind of cell divisions within the seminiferous tubules except an increase in the seminiferous tubules diameter at the expense of cavity and the interstitial space. However, the first spermatogonial division was started at the 5th month of age in Najdi and at 6th months of age in Naemi ram lambs, and spermatogenesis was completed with many spermatozoa occurred at presented the 8th month of age in Najdi and the 9th month of age in Naemi ram. This data showed that the Najdi and Naemi ram lambs reached sings of around puberty the 8th and the 9th months of age Respectively. Sexual development and speed appearance of spermatozoa in ejaculates are more correlated with body weight than chronological age of ram lambs (Toe et al., 2000; Salhab et al., 2002; Lunstra and Cundiff, 2003; Price et al., 2000). Tanyang breed reached puberty between 8th and 9th months of age (Cui et al, 2003), while other breed such as Chios, Karagouniki, Serres, Friesland breeds’ rams reached puberty between 6th and 7th months of age. (Belibasaki and Kouimtzis, 2000; Cui et al., 2003). However, Najdi and Naemi breeds may be among the breeds that are intrinsically of late puberty and this could be a subject of further study.
The current findings indicate that puberty in Najdi and Naemi rams occurs in the 8th and 9th months of age, respectively.
This work was funded by a grant from the Deanship of Scientific Research at King Saud University Riyadh, Saudi Arabia, which is gratefully acknowledged.
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