The effect of varying concentrations of chicken plasma egg yolk and glycerol on the viability of canine sperm following short and long-term preservation.

Background: Plasma egg yolk (PEY), due to simple preparation and easier access, could be a suitable alternative to raw egg yolk for preserving canine semen. Aims: The present study investigated suitable concentrations of PEY and glycerol for preservation of canine semen. Methods: Semen was collected by digital manipulation (seven replicates from four dogs). Following initial raw semen evaluation, the semen was diluted in a tris-based extender supplemented with varying concentrations of chicken PEY (0, 20, and 40% v/v) and glycerol (3%; v/v). After cooling the specimen to 4°C within 1 h, the specimens were diluted with an equal volume of freezing extender consisting of similar concentrations of chicken PEY and 0 and 7% glycerol to reach the final glycerol concentration of 1.5 and 5% for short-term storage of canine semen. Samples with different concentrations of PEY and 5% glycerol were frozen. The sperm viability parameters including total motility, progressive forward motility, plasma membrane integrity, and live percentage of sperm were assessed following short and long-term storage. Results: Sperm viability parameters of semen extended in an extender supplemented with 20 or 40% chicken PEY with either 1.5 or 5% glycerol remained superior until 72 h after semen collection compared to the specimen that did not receive any PEY (P<0.05). Post-thaw sperm viability was also greater in samples extended in extender supplemented with either 20 or 40% PEY compared to 0% PEY. Conclusion: Tris-based extender supplemented with either 20% chicken PEY could be suitable for short and long-term preservation of canine semen.

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 pre-milking 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.

Effect of donor cell age on development of ovine nuclear transfer embryos in vitro.

The effects of the age of cell donor animal on in vitro development of ovine nuclear transfer (NT) embryos were investigated. Somatic donor cells were obtained from two different sources: (1) adult cells (adult fibroblast cells; AFC and adult cumulus cells; ACC); and (2) fetal fibroblasts (40-day-old; FFC-40 and 65-day-old; FFC-65). The fibroblast cell lines were used for NT procedures within 4-13 subpassages. While the cumulus cells were used as non-cultured (fresh) cells. The in vitro matured abattoir-derived oocytes were considered as recipients. No differences in the rates of fusion (75.7, 77.7, 76.3 and 86.7%) and cleavage (80.1, 84.3, 77.8 and 74%) were detected among couplets reconstructed with FFC-40, FFC-65, AFC and ACC, respectively. Blastocyst formation rate of those oocytes reconstructed with FFC-40 was higher (18%; p < 0.001) than those reconstructed with FFC-65 (13%) and AFC (10.9) and comparable with those reconstructed with ACC (17.5%). When the effect of passage number was analysed within groups (FFC-40, FFC-65 and AFC) there were no significant differences in fusion, cleavage and blastocyst rates between reconstructed oocytes. The present study demonstrates that the fetal and adult fibroblasts as well as fresh cumulus cells are comparable in their ability to attain cell fusion and embryonic cleavage. Moreover, the blastocyst formation rate is influenced by the age of the donor animal and the fresh cumulus cells have similar remodelling potential to that of fetal fibroblasts in term of blastocyst formation rate.

Effects of different concentrations of bovine follicular fluid and estrous cow serum on development of murine 2-cell embryos.

Murine 2-cells embryos were isolated from murine oviducts at laboratory and transferred into Ham's F-10 medium containing 0.1 mg mL(-1) streptomycin and 100 IU mL(-1) penicillin G and supplemented with 3 mg mL(-1) bovine serum albumin (BSA) or different concentrations of bovine follicular fluid (bFF) and estrous cow serum (ECS). Significantly higher (p<0.05) > or =4-cell embryos were developed when embryos were cultured 20% bFF (84.33%) comparing to 10 and 15% bFF (48.33 and 69.33%) as well as 3 mg mL(-1) BSA (65.66%). Morula rates were also lower in 10% bFF (22.33%) comparing to the other groups and were similar in 15 and 20% bFF (62.66 and 72.33% morula rates) as well as BSA containing media (55.33%). The highest (p<0.05) blastocyst rates were obtained in medium containing 20% bFF (64.33%) and the lowest belonged to 10% bFF (15%) comparing to 15% bFF (33.66%) or 3 mg mL(-1) BSA. When embryos were cultured in ECS, no significant different was observed in different culture media (76.66, 72.33, 82.5 and 65.66% > or =4-cell embryos in 10, 15 and 20% bFF and 3 mg mL(-1) BSA, respectively). Morula and blastocyst rates were also similar in all groups (32.33, 41.66 and 66.25 and 55.33% morula rates and 15.33, 27, 44.50 and 29.66% blastocyst rates for 10, 15 and 20% bFF and 3 mg mL(-1) BSA, respectively). The results of the present study demonstrated that 20% bFF could be substituted for BSA when in vitro culture of murine embryos is carried.

Maintenance of pregnancy in repeat breeder dairy cows by CIDR administration after breeding.

The aim of this study was to assess the effect of two periods of P4 therapy by CIDR on the 1st service Conception Rate (CR) of repeat breeder dairy cows. The cows were selected on the following basis: absence of dystocia, retained placenta and endometritis after the last parturition. 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 day 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 45-55 days after AI in cows not observed in estrus. The difference in CR among the groups was analyzed by chi square analysis. The CR in groups A, B and C was 55, 41.7 and 30%, respectively and was greater (p<0.05) in treatment (A) cows than in control cows. 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 treatment period (4 days treatment).

Pregnancy rate following post-insemination intrauterine treatment of endometritis in dairy cattle.

Summary Of 1500 examined Holstein cows in milk, there were 315 cows with mucopurulent discharges at the time of insemination (21%). Twenty-four hours after insemination, the infected animals randomly received no treatment (group I, control, n = 93), intrauterine infusion of 1 g oxytetracycline (group II, n = 63) and intrauterine infusion of 5 million IU procain penicillin G sodium (group III, n = 159). First service conception rate following treatment was 48.3, 49.2 and 47.7% in groups I, II and III, respectively. In conclusion, treatment with either antibiotic had no advantage relative to the control on first service conception rate.

Ultrasonic characteristics of preovulatory follicle and ovulation in Caspian mares.

The Caspian breed of horses is believed to be the direct descendant of the earliest equine animals. Some special characteristics of Caspian horse differentiate this breed of horses from other breeds. In the current study the ultrasonically observed characteristics of a preovulatory dominant follicle and the lengths of estrus, diestrus as well as some related parameters were studied during 42 interovulatory intervals in 11 healthy Caspian mares. The preovulatory dominant follicle deviated from subordinate follicles and became the largest follicle in the ovaries at Day -8.7 +/- 0.53 (Day 0=ovulation). Every mare was a single ovulator with ovulations more frequent from the left ovary than from the right (65% versus 35%). Mean length of estrus, diestrus, and interovulatory interval were 8.3 +/- 0.86, 13.8 +/- 0.59, and 22.1 +/- 0.40 days, respectively. The time interval from ovulation until the time in which the mares were no longer in estrus was 1.9 +/- 0.42 days.