[Effect of vitamin C on colony formation of ovine spermatogonial stem cells in vitro]

Background and Aim: The technology of using spermatogonial stem cells [SSCs] has been limited due to lack of an ideal culture system for growth and proliferation. The aim of this study was to investigate the effects of different doses of vitamin C on SSCs colony formation in vitro

Materials and Methods: The cells were isolated from testes of prepubertal lambs by two enzymatic digestions, purified by differential platting, and then treated for 10 days by using 4 methods: Simple culture including SSCs in DMEM containing 1% antibiotic and 5% FBS as our control group and for the three other cultures we used the same culture medium as that in control group plus 20, 40 and 60 micro g/ml of vitamin C respectively. Culture media were refreshed every 72h and colony numbers and diameters were determined on the 4[th], 7[th] and 10[th] days after the beginning of culture by using inverted microscope. Spermatogonial cells were identified by immunocytochemistry staining against PGP9.5. Using R software, the results obtained from 5 repeats were evaluated by ANOVA. P<0.05 was considered significant

Results: On the 7[th] day, we found a significant difference between the culture No. 2 [0.41 mm[2]] and culture No. 3 [0.08 mm[2]] in regard to spermatogonial colonies surface areas [P<0.05]. Also, colonies surface areas on the 10[th] day in the culture No. 2 was significantly greater than those in the other groups [P<0.05]

Conclusion: The results of this study showed that vitamin C with a dose of 40 [micro g/ml] was effective in increasing the surface area of spermatogonial colonies. But it had no effect on spermatogonial cell number

case of perosomus elumbis concurrent with visceral abnormalities in a Holstein calf

Perosomus elumbis is an occasional congenital anomaly of cattle, swine, sheep, and dogs with unknown etiology. This congenital abnormality occurs in both sexes. A dead Holstein calf characterized by musculoskeletal and external genitalia abnormalities was referred to the large animal hospital of University of Tehran. Radiographic evaluation and subsequent dissection revealed that the vertebral column was truncated at the level of first lumbar vertebra [L1]. Moreover, L2-L5, sacrum and coccygeal vertebrae were absent. The dorsum of the lumbosacral region contained only soft tissues. Urogenital tract was incomplete, and it contained agenesis of the ovaries, uterine tubes, cervix, and vulva concurrent with unilateral umbilical artery agenesis. Small and large intestine contained blind-ended sacs. No testes, scrotum, and penis were found. The intact ureter was attached to a thin-walled fluid fill sac. The laboratory finding showed that the pH of the fluid was 6 and contained hemoglobin, white blood cells, bacteria, a few red blood cells, oxalate crystalline, and epithelial cells. It was concluded that the collected fluid was urine. This is the first report of perosomus elumbis in a Holstein calf having a lot of visceral abnormalities in Iran

Isolation of bovine spermatogonial cells and co-culture with prepubertal sertoli cells in the presence of colony stimulating factor-1

Spermatogonial stem cells [SSCs] are infrequent self-renewing cells among the type A spermatogonia within the seminiferous tubules and are the basis of spermatogenesis in mammalian testis. An adequate number of SSCs is a primary requirement for the study of their behavior, regulation, and further biomanipulation. In this paper, we studied the development of the primary co-cultures of type A spermatogonia and prepubertal bovine sertoli cells in the presence of Colony Stimulating Factor 1 [CSF1], a potential contributor in the SSC niche. The effect of different concentrations of CSF1 [0, 10, 50 and 100 ng/mL] on the colonization activity of spermatogonial cells was assessed 4, 7 and 11 days after the beginning of the culture by counting the total number of colonies and measuring their area in each group of the present experiment. Immunofluorescent staining against OCT4 and vimentin led to the confirmation of the nature of both the SSCs and sertoli cells. Results showed that the total number of colonies from day 4 to 11 increased significantly in all groups, independent of CSF1 concentration. In addition, the total number and total area of colonies were higher [not significant] in 10 and 50 ng/mL CSF1 treatments than the control and 100 ng/mL CSF1 groups in all the three evaluations during the experiment. However, this difference was only significant [p<0.05] between the total area of colonies in the control and 10 ng/mLCSF1 groups at day 4 of co-culture. It was concluded that CSF1 can be a suitable growth factor for improving SSCs colonization in vitro, particularly during the first days of culture where accompanying sertoli cells still have not proliferated sufficiently to support the propagating spermatogonial cells

[Comparison between progesterone and GnRH supplementation on the conception rate of heat stressed dairy cattle after artificial insemination]

Heat stress causes reduced fertility and significant economic loss in dairy cattle. To override the suppressive effects of heat stress, various hormonal manipulations have been utilized. The aim of this study was to compare the effect of progesterone [in the form of CIDR] and administration of GnRH after insemination on the conception rate of heat stressed dairy cattle. All cows were inseminated at estrus and were then alternately assigned into three groups on day 5 after artificial insemination [AI]: i] GnRH group [n=44] received an IM injection of 500 micro g GnRH [GONAbreed, PARNEL, Australia,]; ii] CIDR group [n = 44] received a CIDR [EAZI-BREED, Hamilton, NZ, containing 1/9 g progesterone] which was removed after a week; and iii] control group [n = 36], which did not receive any treatment. Conception was diagnosed on day 32-39 after AI by ultrasonography. Conception rate in GnRH, CIDR and control groups were 54.5%, 54.5% and 58.3%, respectively. The results demonstrated that there was no significant difference among the three groups [p >0.05]. These treatments had had no statistically different effects on lactation, milk yield, days in milk and number of AI [p>0.05]. Conception rates within GnRH and CIDR groups in <150 and >150 days in milk subgroups were 74.4%, 40.7%, 84.6% and 41.9%, respectively and differed statistically significantly [p>0.05]. Conception rate within control and CIDR groups among <3 and >3 numbers of AI were 80%, 31.2%, 84.2% and 32%, respectively, which was statistically significant [p>0.05]. According to the results of this study, the use of GnRH and CIDR after AI did not improve conception rates of mildly heat stressed dairy cattle