[Transfection of bovine spermatogonial stem cells in vitro]

Spermatogonial stem cells [SSCs] are the only stem cells in adults that can transfer genetic information to the future generations. Considering the fact that a single SSC gives rise to a vast number of spermatozoa, genetic manipulation of these cells is a potential novel technology with feasible application to various animal species. The aim of this study was to evaluate enhanced green fluorescent protein [EGFP] gene transfection into bovine SSCs via liposome carrier and assess the best incubation day in uptake exogenous gene by SSCs. Transfection efficiency of EGFP gene with lipofectamine 2000 was determined in days following each three day of transfection [day 4, 6 and 8 of the culture] by fluorescent microscope. Results showed that the transfected cells through lipofection increased significantly [P<0.05] in each three days of transfection in comparison with those of the control groups. The transfected SSCs were higher in comparison with those of the free exogenous gene carrier groups [P<0.05]. In comparison with these three days, the rate of infected cells was higher when transfection proceeds at day four. It was concluded that lipofectamine can be used safely for direct loading exogenous DNA to SSCs particularly during the fourth day of culture

Ultrasonography of the supramammary lymph nodes for diagnosis of bovine chronic subclinical mastitis

Currently, somatic cell count [SCC] and bacterial culture are considered as the gold standard of detecting subclinical Mastitis. Mastitis leads to proliferation of lymphocytes in the supramammary lymph nodes and subsequent enlargement of ipsilateral lymph node. Ultrasonography can be used to survey these changes. A portable ultrasound machine with a 2-5 MHz convex transducer was used to identify the supramammary lymph node size in 35 cows in a herd with chronic Staphylococcus aureus mastitis. After premilking udder preparation, a California mastitis test [CMT] was performed and individual milk samples were taken from each quarter for bacterial culture and somatic cell count [SCC] in accordance with NMC recommendations. The mean length [range 5.77-12.90 cm] and width [range 2.07-7.41 cm] of the lymph node were 9.2 and 4.03 cm, respectively. There was a positive correlation between lymph node size [length and depth] and culture of milk samples on ipsilateral quarters. Also, there was a significant difference correlation between CMT or mean log SCC of each side and size of supramammary lymph node in the same side. This study showed significant changes in supramammary lymph node dimensions in mastitis cases, so ultrasonography of this lymph node is probably a useful method for mastitis detection, especially in situations that test on milk is impossible

Evaluation of therapeutic effects of a 5-day intrauterine infusion of 6% Eucalyptus globulus oil solution on mares with experimentally induced endometritis with Streptococcus zooepidemicus

Endometritis is one of the major causes of infertility in mares. The present study was conducted to evaluate intrauterine infusion of Eucalyptus globulus oil as treatment for mares with experimentally induced endometritis. Mares [n=8] were synchronized using two injections of PGF 2alpha [10 mg]14 days apart. All mares were inoculated with a live culture of 5 × 10[6] CFU S. zooepidemicus in the dioestrus phase after the termination of oestrus. The day of inoculation was considered as day 0 of the experiment. On days 3, 6 and 9, cytological and bacteriological specimens were collected to determine endometritis. Mares were randomly assigned to two groups on day 10. Mares in treatment [n=4] and control [n=4] groups received intrauterine 6% Eucalyptus oil solution [100 ml] and intrauterine saline solution [100 ml] for 5 consecutive days. Cytological, bacteriological, and ultrasonographic evaluations were implemented at days 15, 17 and 19 [1, 3 and 5 days after termination of intrauterine treatment, respectively]. The number of mares diagnosed with endometritis, the depth of their intraluminal uterine fluid and the number of infected mares did not change by the treatment [P>0.05]. In conclusion, the 5-day intrauterine infusion of 6% E. globulus oil solution failed to treat experimental endometritis in mares

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

[Ultrasonographic study on uterine involution following induction of abortion in bitch]

Based on our knowledge, there have been no ultrasonographic studies on uterine involution following induction of abortion in the bitch. To evaluate ultrasonographic features of uterine involution following induction of abortion. In a concurrent study, pregnancy was terminated in 6 bitches in the third trimester of pregnancy by ultrasound guided induced fetal death. Ultrasonographic examination of the reproductive tract was carried out after the day of complete abortion up to complete uterine involution, in which uterine dimensions were unchanged within two consecutive evaluations. Uterine shape, size, and echogenicity as well as wall layering were evaluated in ultrasonographic evaluations, and mean values were measured in both placental and interplacental regions. Ultrasonographic features revealed that, uterine wall is detected on ultrasound images having six different layers on the basis of its echogenicity. Wall layering was very explicit and distinct within the first week after abortion and lost its distinction in the course of uterine involution. On the day after abortion, placental regions measured approximately twice the size of the uterus in interplacental areas. In the first week, the mean values of the placental and interplacental thickness were 2.11 +/- 0.31cm and 1.12 +/- 0.20 cm respectively, measured in uterine horns. After the third week, the placental and interplacental regions lost their distinction and thickness. In the last two weeks, uterine horns were almost equal in dimension in every part. The time of complete involution varied amongst these 6 different bitches, being in the 6[th], 8[th], 9[th], 10[th], and 13[th] week after the induction of abortion. The findings of this study could be a basis for ultrasonographic evaluation of abnormal uterine structures and also detection of uterine involution time after abortion or parturition

Control of red deer [Cervus elaphus] stag domination in rut season by estrus synchronization

Inbreeding is one of the major problems of small size herds of red deer in refuge wildlife parks. The aim of the present study was to use estrus synchronization technique in red deer [Cervus elaphus] based on natural mating to control the dominancy and social rank of stags and inbreeding. In this study, hinds [n=6] were captured and caged at breeding season [early October] in Semeskande Wild Life Refuge Park. The captured hinds were treated with intravaginal progesterone devices [CIDR] for 12 days, followed by injection of 250 IU PMSG at the time of CIDR withdrawal. After 48 hours, all 6 hinds were released from cages. In estrus phase, the chase sequences started and the old dominant stag could not control all of the hinds in his territory so the two other young stags successfully mated with the other hinds. Four out of six hinds became pregnant and the calves were born in early June. The results of this study showed that the estrus synchronization technique in Refuge Wildlife Park which is based on natural mating, can be used successfully to control inbreeding easily and give young stags the opportunity to copulate

[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

Effects of dietary polyunsaturated fatty acids on ovarian function and prostaglandin secretion in lactating dairy cows

As lactating cows in severe negative energy balance have poor reproductive performance, the effect of dietary fat supplementation [fish oil, soybean oil] on PGFM secretion, ovarian function and blood metabolites is investigated. In this experiment, the effects of dietary polyunsaturated fatty acids on plasma metabolites, ovarian function and prostaglandin secretion of 20 primiparous Holstein cows was studied. The cows were randomly allocated to one of four groups that were fed either: 1] a control diet; 2] a diet with 3% [Feed dry matter basis] fish oil; 3] a diet with 3% soybean oil; or 4] a diet with 1.5% fish oil and 1.5% soybean oil. Groups were synchronized using the heat-synch method and were fed their respective diets for 35 days, allowing 14 days for dietary adaptation and 21 days for data collection. Concentration of plasma glucose, triglycerides and low density lipoprotein [LDL] cholesterol were not affected by the treatments, but plasma total cholesterol and high density lipoprotein [HDL] cholesterol concentrations were significantly higher in cows that consumed the oil-containing diets [p < 0.05].The number of follicles, corpus luteum size and plasma estradiol, progesterone and prostaglandin F[2a] metabolite [PGFM] concentrations were similar across all treatments. However, the size of the largest follicle was significantly greater in cows that consumed a diet containing fish oil or soybean oil [p < 0.05]. These results suggest that polyunsaturated fatty acids can influence both ovarian and uterine function in cows, but further studies are required to test their effects on dairy cow reproduction

[ effect of two different periods of CIDR supplementation on the second service conception rate of repear breeder dairy cows]

Repeat breeding [RB] is a syndrome and several factors have been identified as its causes. Luteal insufficiency is a known cause of embryonic mortality. The aim of this study was to assess the effect of two periods of P4 therapy on the Second service and also their effects on the First plus the second service conception rates [CR] of repeat breeder dairy cows. Cows included in the study were in their first to filth lactation and had 3 to 6 unsuccessful inseminations within the current lactation. They were inseminated according to the AM/PM rule relative to estrus onset, and randomly assigned into 3 groups: [A] CIDR on day 5 after insemination that was removed on day 9 of the cycle [n=40]; [B] CIDR on days 5 after insemination that was removed on day 19 of the cycle [n=36]; and [C] untreated controls [n=40]. Pregnancy diagnosis was conducted by rectal palpation about 45 days after Al in cows no observed in estrus. The second service CR and overall CR in groups A, B and C were 61.7, 42.9.21.4 and 82.5, 66.7, 45 percent, respectively. Group A showed higher level compared to the controls [p<0.05]. In conclusion, repeat breeder cows in groups A and B benefited from progesterone supplementation, but significant effects of treatment for improvement of conception rate was seen in short term treatment [4 days treatment]

influence of foal heat oxytocin injection with or without lunging in foal heat on conception rate in mares with intrauterine fluid accumulation

To study the effect of oxytocin injection and also oxytocin plus lunging on intrauterine fluid reduction and conception rate in mares with intrauterine fluid accumulation. Experimental study. A total of 45 mares, 8-14 years old. Among the mares in foal heat, 45 mares with more than 1 cm2 fluid in their uterus [evaluated by ultrasonography] were selected and categorized randomly in 3 groups as follow: Group 1: Without treatment [control]. N=17. Group 2: Oxytocin injection [15 iu, IV], one daily, up to the end of foal heat [first injection on the 2[nd]day of foal heat]. N=14. Group 3: Treatment as group 2 plus lunging for 20-30 minutes, initiated 60 minutes after oxytocin injection [to evaluate the effect of lunging in association with oxytocin on uterine drainage]. N=14 Results: Reduction of uterine fluid after treatment was 71.4% and 98.9% in group 2 and 3 respectively compared with 17.6% in control group [P<0.05]. The total conception rate in 3 groups with uterine fluid less than 1 cm[2][61.5%] was significantly more than the total one in 3 groups with uterine fluid > 1 cm[2][26.3%][P<0.05]. Lunging and or oxytocin injection in mares with intrauterine fluid accumulation in foal heat can improve conception rate in next estrus

In vitro maturation of dromedary camel oocytes in Hamsvs F10 medium supplemented with different concentrations of fetal bovine serum

To mature dromedary camel oocytes for using them in an IVF system. Design: Interventional study. Ovaries from dromedary camels in local slaughterhouses. Removing varies from camels in a local slaughterhouse, carrying them to the laboratory in warm saline solution, aspiration of follicles, isolation and transferring of oocytes into TCM-199 and Ham's F10 supplemented with 0-10% heat inactivated fetal bovine serum [FBS], culturing oocytes for up to 24h in a COz incubator. After culture oocytes were denuded and put into PBS containing 0.1% hyaluronidase and passing through a fine pipette. Oocytes were then mounted onto slide glass and fixed and stained for evidence of maturation. ANOVA and when a significance different was seen, Duncan's Multiple Range Test. When oocytes from fresh ovaries were culture in Ham's F10 without protein, only 17.65% of them reached to MIL However, significantly [P<0.05] higher oocytes reached to Mil in 5 and 10% PCS [36.84% and 33.33% for 5 and 10% FBS respectively], which were not dose dependent. When cool stored ovaries were used for oocyte maturation, 14.54% of oocytes reached to MIL In protein-free medium However, significantly [P<0.05] higher oocytes reached to Mil in 5 and 10% PCS [25.86% and 33.33% for 5 and 10% FBS respectively]. Although increasing the protein increased the maturation rates, the difference was not significant. Under the present condition it seems that cool stored ovaries could be used for in vitro maturation of camel oocytes