Effect of follicular ablation and gonadotropin priming on the recovery and quality of oocytes in Boran cows.

The objective of the study was to evaluate the effect of follicle ablation, exogenous FSH application, and different coasting time prior to ovum pick-up (OPU) on the number of follicles suitable for aspiration, oocyte quality, and cleavage rate in Ethiopian Boran cows. The experiment was carried out in three parts, (I) cows were synchronized using 500 µg PGF2α given 11 days apart. Cows were then subjected to a biweekly ovum pickup session before ovulation (n = 5) or starting day 7 after ovulation (n = 4) for 3 weeks. (II) Cows were synchronized, and all visible follicles were ablated on the first days of overt estrus, and cows were grouped into those that received a divided dose of 350 IU FSH (n = 5) or 175 IU FSH (n = 5) over 3 days. In both groups, OPU was carried out weekly starting 48 h after the last FSH for 6 weeks. (III) Protocol was similar to part II, but in group with 350 IU FSH (n = 5), coasting period was increased to 72 h. The covariates of follicles and oocyte were not affected (P > 0.05) by corpus luteum presence at OPU. The mean number of medium (7.36 ± 0.57) and large (8.28 ± 0.96) follicles were significantly higher (P < 0.05) in the group that received divided 350 IU FSH. Similarly, the mean number of grade-1 (4.19 ± 0.24) and grade-2 (4.32 ± .27) oocytes, maturation rate (70.41%), and cleavage rate (47.5%) were significantly higher (P < 0.05) in the group that received 350 IU FSH. COC quality was significantly (P < 0.05) influenced by coasting period. However, both maturation and cleavage rates were not affected by the coasting period. This study demonstrated that follicular ablation and treatment with FSH improves follicular population and oocyte recovery rate in Boran cows.

Influence of Nano-Methionine supplementation in drinking water on growth performance, lipid metabolism, and related gene expression in broiler chicken.

Objectives: The study aimed to determine how Nano-Methionine (Nano-Meth) affected growth, lipid metabolism, and relative gene expression for acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), growth hormone receptor (GHR), insulin-like growth factor receptor-1 (IGFR-1), myostatin (MSTN), and cholecystokinin (CCK) genes in broiler chickens. Materials and Methods: A total of 100 1-day-old broilers were randomly assigned into 2 groups: 1) the control group received drinking water without any supplements, and 2) the Nano-Meth group received 10 ml/l of 5% Nano-Meth starting from 1 day old until 35 days old (the end of the experiment). Results: Nano-Meth improved final body weight, weight gains, feed intake, and feed conversion ratio. Compared to the control group, Nano-Meth significantly lowered the serum levels of triglyceride, cholesterol, very low-density lipoprotein, and low-density lipoprotein in chickens. Nano-Meth significantly increased the serum levels of total protein, albumin, high-density lipoprotein, and glucose more than the control group. Nano-Meth lowered the mRNA gene expression of ACC, FAS, MSTN, and CCK but increased that of GHR and IGFR-1. Conclusions: We concluded that supplementation with Nano-Meth enhances growth performance and decreases lipid accumulation in broiler chickens.

Dietary supplementation of nano-selenium eliminates the negative effects of long-term ivermectin injection on growth and reproductive performance of female rabbits.

Objective: We investigated the effects of a source of selenium [inorganic or nano-selenium (nano-Se)] on female V-line rabbits with or without injection of ivermectin (IVM). Material and Methods: Eighty four rabbits (12 weeks old) were randomly divided into 4 groups of 21 rabbits each with the basal diet supplemented as per the following treatments: G1 (control): inorganic Se at 0.3 mg/kg diet with no IVM injection; G2: inorganic Se with IVM injection; G3: nano-Se with no IVM injection; and G4: nano-Se with IVM injection. IVM was injected subcutaneously at 0.2 mg/kg body weight starting when the does were 14 weeks old and repeated weekly for five consecutive weeks. Results: Replacement of inorganic Se with nano-Se improved body weight and total body weight gain, total feed intake, average feed conversion ratio, and reproductive performance (age at puberty, number of service/conception, conception rate, number of kits/litter, and litter weight at birth). Similarly, sexual activity of does, serum estrogen levels, and serum levels of antioxidants (glutathione reduced, catalase, and malondialdehyde) increased in nano-Se-supplemented groups. Ivermectin treatment in inorganic Se-supplemented groups was detrimental to growth and reproductive performance, while these parameters improved in IVM-treated and nano-Se-supplemented groups. Conclusion: Nano-Se mitigated the negative effects of IVM treatment on the growth and reproductive performance of does.

Effect of cryopreservation and in vitro culture of bovine fibroblasts on histone acetylation levels and in vitro development of hand-made cloned embryos.

In this study, the relative acetylation levels of histone 3 in lysine 9 (H3K9ac) in cultured and cryopreserved bovine fibroblasts was measured and we determined the influence of the epigenetic status of three cultured (C1, C2 and C3) donor cell lines on the in vitro development of reconstructed bovine embryos. Results showed that cryopreservation did not alter the overall acetylation levels of H3K9 in bovine fibroblasts analysed immediately after thawing (frozen/thawed) compared with fibroblasts cultured for a period of time after thawing. However, reduced cleavage rates were noted in embryos reconstructed with fibroblasts used immediately after thawing. Cell passage affects the levels of H3K9ac in bovine fibroblasts, decreasing after P1 and donor cells with lower H3K9ac produced a greater frequency of embryo development to the blastocyst stage. Cryopreservation did not influence the total cell and ICM numbers, or the ICM/TPD ratios of reconstructed embryos. However, the genetic source of donor cells did influence the total number of cells and the trophectoderm cell numbers, and the cell passage influenced the total ICM cell numbers.

Intraocular pressure in captive black-footed penguins (Spheniscus demevsus) measured by rebound tonometry.

Intraocular pressure (IOP) measurement is a common procedure during eye examinations in birds. Differences in the IOP between avian species have been reported, which suggests the need to establish species-specific reference ranges. To determine IOP values of captive black-footed penguins (Spheniscus demersus), we obtained IOP readings with the use of a rebound tonometer by using two established calibration settings (dog and horse). No difference was seen in the IOP between the left and right eye when the horse setting was used; however, a difference was present when using the dog setting. No significant difference between the IOP of male and female penguins was seen in both eyes when the dog or horse setting was used. Rebound tonometry appears to be a safe and repeatable method to obtain IOP values in black-footed penguins.

Dietary supplementation of nano-selenium improves reproductive performance, sexual behavior and deposition of selenium in the testis and ovary of Japanese quail.

OBJECTIVE: Selenium (Se), as the form of selenite, is commonly supplemented in poultry diet, which has low bioavailability and high toxicity. Here, we compared the effects of the supplementation of the diet with Se nanoparticles (nano-Se) on the growth, sexual behavior, and reproductive performance (gonad size, sperm quality traits, and plasma testosterone levels for males and egg production for females) of Japanese quail (Coturnix coturnix japonica). MATERIALS AND METHODS: Quail chicks (n = 300) aging 14 days were divided into three groups: Group 1 (basal diet and Se at 0.2 mg/kg ration), Group 2 (basal diet and nano-Se at 0.2 mg/kg ration), and Group 3 (basal diet and nano-Se at 0.1 mg/kg ration). Several parameters relating to body weight and egg were measured. Sexual behaviors of the birds were observed by continuous visual scanning. The sperm viability, sperm morphology, and concentration of spermatozoa were determined after staining and microscopic examination. The plasma testosterone levels were determined by indirect enzyme immunoassay assay. The Se concentrations in the testicular, ovarian, and ration samples were measured by flame emission atomic absorption spectrophotometer. RESULTS: The type or concentration of nano-Se administration had no impact on body weight, feed efficiency, egg production, or egg weight. However, the total feed intake throughout the experiment was reduced in Group 2 at 0.2 mg/kg. Nano-Se supplementation significantly increased the sexual behavior. In general, the deposition of Se in the testicular and ovarian tissues increased with increasing supplement concentration. At the same supplement concentration, the tissue deposition of nano-Se was more enhanced than that of inorganic Se. Nano-Se supplementation improved the testicular functions by enhancing plasma testosterone level and sperm quality traits (sperm count, motility, and viability). This improvement was found more prominent with the lower supplement concentration (when comparing 0.1 vs. 0.2 mg/kg diet). CONCLUSION: It is concluded that the use of nano-Se (at 0.1 mg/kg) in the ration of Japanese quail improves several reproductive performance parameters.

Production of bovine cloned embryos with donor cells frozen at a slow cooling rate in a conventional freezer (-20 degrees C).

SummaryUsually, fibroblasts are frozen in dimethyl sulphoxide (DMSO, 10% v/v) at a cooling rate of 1 degrees C/min in a low-temperature (-80 degrees C) freezer (LTF) before storage in liquid nitrogen (LN2); however, a LTF is not always available. The purpose of the present study was to evaluate apoptosis and viability of bovine fibroblasts frozen in a LTF or conventional freezer (CF; -20 degrees C) and their subsequent ability for development to blastocyst stage after fusion with enucleated bovine oocytes. Percentages of live cells frozen in LTF (49.5%) and CF (50.6%) were similar, but significantly less than non-frozen control (88%). In both CF and LTF, percentages of live apoptotic cells exposed to LN2 after freezing were lower (4% and 5%, respectively) as compared with unexposed cells (10% and 18%, respectively). Cells frozen in a CF had fewer cell doublings/24 h (0.45) and required more days (9.1) to reach 100% confluence at the first passage (P) after thawing and plating as compared with cells frozen in a LTF (0.96 and 4.0 days, respectively). Hypoploidy at P12 was higher than at P4 in cells frozen in either a CF (37.5% vs. 19.2%) or in a LTF (30.0% vs. 15.4%). A second-generation cryo-solution reduced the incidence of necrosis (29.4%) at 0 h after thawing as compared with that of a first generation cryo-solution (DMEM + DMSO, 60.2%). The percentage of apoptosis in live cells was affected by cooling rate (CF = 1.9% vs. LFT = 0.7%). Development of bovine cloned embryos to the blastocyst stage was not affected by cooling rate or freezer type.

Cloned embryos from semen. Part 2: intergeneric nuclear transfer of semen-derived eland (Taurotragus oryx) epithelial cells into bovine oocytes.

The production of cloned offspring by nuclear transfer (NT) of semen-derived somatic cells holds considerable potential for the incorporation of novel genes into endangered species populations. Because oocytes from endangered species are scarce, domestic species oocytes are often used as cytoplasts for interspecies NT. In the present study, epithelial cells isolated from eland semen were used for intergeneric transfer (IgNT) into enucleated bovine oocytes and compared with bovine NT embryos. Cleavage rates of bovine NT and eland IgNT embryos were similar (80 vs. 83%, respectively; p > 0.05); however, development to the morula and blastocyst stage was higher for bovine NT embryos (38 and 21%, respectively; p < 0.0001), than for eland IgNT embryos (0.5 and 0%, respectively). DNA synthesis was not observed in either bovine NT or eland IgNT cybrids before activation, but in 75 and 70% of bovine NT and eland igNT embryos, respectively, cell-cycle resumption was observed at 16 h postactivation (hpa). For eland IgNT embryos, 13% had > or = 8 cells at 84 hpa, while 32% of the bovine NT embryos had > or = 8 cells at the same interval. However, 100 and 66% of bovine NT and eland IgNT embryos, respectively, that had > or = 8 cells synthesized DNA. From these results we concluded that (1) semen-derived epithelial cell nuclei can interact and be transcriptionally controlled by bovine cytoplast, (2) the first cell-cycle occurred in IgNT embryos, (3) a high frequency of developmental arrest occurs before the eight-cell stage in IgNT embryos, and (4) IgNT embryos that progress through the early cleavage stage arrest can (a) synthesize DNA, (b) progress through subsequent cell cycles, and (c) may have the potential to develop further.

Cloned embryos from semen. Part 1: in vitro proliferation of epithelial cells on embryonic fibroblasts after isolation from semen by gradient centrifugation.

Although epithelial-like somatic cells have been previously isolated from semen, cell proliferation rates were low. Culture of whole semen samples resulted in loss of potentially valuable spermatozoa. The aims of the present study were to: (1) isolate somatic cells from semen, while preserving sperm viability, and (2) optimize in vitro culture conditions for semen-derived epithelial cells. Density gradient centrifugation of washed ejaculates of two rams (Ovis aries) (n = 24) and one eland bull (Taurotragus oryx) (n = 4) was performed using a three-layer discontinuous Percoll column consisting of 90% (P-90), 50% (P-50), and 20% (P-20) Percoll. In vitro culture and Trypan Blue staining indicated that live somatic cells settled in the P-20 layer. Nonmotile spermatozoa were recovered at the P-50 and P-90 interfaces, whereas motile spermatozoa were collected in the pellet from the P-90 layer. Subsequently, somatic cells isolated from the P-20 layer were plated either on inactivated 3T3 mouse embryonic fibroblast feeder layers, collagen-coated plates with 3T3 feeder cell inserts, or on collagen-coated plates. Initial somatic cell plating was similar among treatments, but proliferation significantly increased when cocultured with 3T3 cells (feeder or insert). Furthermore, two different types of epithelial cells were obtained. The exact origin of the cells in the male reproduction system is uncertain and probably variable. The present method of cell isolation and in vitro culture may be of value for preserving endangered species. Specifically, cells isolated and cultured from cryopreserved semen of nonliving males could be used for producing embryos by somatic cell nuclear transfer.

Reversal of motility loss in bongo antelope (Tragelaphus eurycerus isaaci) spermatozoa contaminated with hyposmotic urine during electroejaculation.

Semen collected by a combination of ampullary (rectal) massage and electroejaculation of a bongo bull was incidentally contaminated with urine (1:3.7). At 1.5h post-collection, progressive motility was 0% but some spermatozoa had intermittently twitching tails. Subsequent dilution with media and processing improved the progressive motility (up to 50%) and intact membranes (up to 71%) of spermatozoa. After thawing, the respective values were 35 and 70%. The osmolarity and pH of the contaminated supernatant was 151 mOsm and 7.45, respectively. Initial progressive motility in a non-contaminated portion of semen collected during the same procedure was 80%, and, after thawing, 60 and 90%, of the spermatozoa showed progressive motility and intact membranes, respectively. In conclusion, urine-contaminated bongo spermatozoa can regain progressive motility after dilution with isosmotic solutions and survive cryopreservation.

Behavioral training and hydraulic chute restraint enables handling of eland antelope (Taurotragus oryx) without general anesthesia.

Difficulties and risks associated with restraining large nondomestic ungulates are limiting factors toward developing and applying assisted reproductive technologies, such as artificial insemination and embryo transfer. In this study on 10 female eland (Taurotragus oryx), we evaluated the use of behavioral training and handling handling in a hydraulic chute to perform transvaginal ultrasound-guided oocyte retrieval and other clinical procedures. Nine females were conditioned to associate specific sound cues with food treats. The interval from the audio cue until acceptance of handheld treats varied among females (1.8-58.3 min). Animals also differed in their response to training for voluntary entry into the chute. Handling eland for oocyte retrieval in the hydraulic chute required xylazine sedation. During sedation and handling, eland undergoing oocyte retrieval procedures had higher blood glucose levels (14.4 +/- 3.1) than females handled similarly but without oocyte retrieval (9.3 +/- 2.7 mmol/L). Plasma osmotic pressure, hematocrit, and creatine phosphokinase activity were similar between these two groups. Females that were more difficult to train had higher blood glucose levels than the more cooperative animals. Cooperative females had fewer vertical stripes on their sides. More than 40 procedures were conducted without complications or mortality. The combination of behavioral conditioning-training and restraint of sedated eland in a hydraulic chute was a reliable and repeatable method for performing minimally invasive assisted reproductive techniques.

Blastocyst development after intergeneric nuclear transfer of mountain bongo antelope somatic cells into bovine oocytes.

Intergeneric embryos were constructed by nuclear transfer using Mountain Bongo antelope somatic cells fused with enucleated bovine oocytes and their subsequent development in vitro was investigated. After two to six passages, starved or non-starved skin fibroblast cells were used as donor nuclei. In vitro matured bovine oocytes were enucleated by squeezing the first polar body and surrounding cytoplasm through a slit in the zona pellucida. After injection of a somatic cell into the perivitelline space, couplets were fused electrically and activated chemically, then subjected to different embryo culture treatments. Serum starvation had no effect on the frequency of cleavage to two cells or on development to the blastocyst stage in either sequential hamster embryo culture medium (HECM)-6/TCM-199 + serum or HECM-9/TC-199 + serum, or modified synthetic oviduct fluid (mSOF) culture medium. When couplets from non-starved donor nuclei were cultured, the frequency of cleavage (66 +/- 8% vs. 44 +/- 5%), development to >/=9 cells (46 +/- 6% vs. 24 +/- 4%), and formation of blastocysts (24 +/- 5% vs. 11 +/- 2%) were all significantly higher (p < 0.05) in the HECM-6 medium than in mSOF medium. In conclusion, bovine oocytes can support blastocyst development after intergeneric fusion with bongo fibroblasts. This technique could potentially be used as an alternative to using scarce bongo oocytes in attempts to propagate these endangered animals.

Ultrasonographic-guided retrieval and in vitro maturation of eland (Taurotragus oryx) and bongo (Tragelaphus eurycerus isaaci) antelope oocytes.

The limited availability of gametes is a major factor hindering the development and application of assisted reproductive technologies (ART) in large non-domestic ungulates. This is partly due to the small number of captive animals and handling difficulties associated with procedures for gamete recovery. In the present study, results are reported of multi-year studies on ovarian stimulation and oocyte retrieval by ultrasonographic-guided transvaginal follicular aspiration and subsequent in vitro maturation (IVM) in eland and bongo antelopes. All procedures were conducted on sedated females handled in a hydraulic chute without inducing general anesthesia. Five estrous synchronization/ovarian stimulation protocols were evaluated and data are presented on 73 and 15 procedures in eland and bongo, respectively. Repeating procedures (< or =once/month) on the same female did not affect ovarian response or number oocytes recovered in either species. Eland females, but not the ovarian stimulation treatment, affected ovarian response. Ovarian stimulation treatment affected oocyte recovery rate in eland, but not in bongo. In both species, ovarian hormone stimulation treatment affected the distribution of follicles by size and the status of expansion of the cumulus cell investment of oocytes, but not the frequency of metaphase II oocytes during IVM. The timing of extrusion of the first polar body during IVM was more synchronous in bongo than in eland oocytes. It is concluded that Transvaginal oocyte retrieval (TVOR) can be safely and repeatedly applied in gonadotropin-treated eland and bongo females to recover oocytes that can mature in vitro. The methods described for the present study can be adapted to improve the availability of non-domestic ungulate oocytes for basic and applied studies.