Isolation and Culture of Ovine and Bubaline Small and Large Pre-antral Follicles: Effect of Cyclicity and Presence of a Dominant Follicle

Studies were conducted to examine the effects of the cyclicity and the presence of a dominant follicle (DF) in ovary on the recovery and in vitro growth of pre-antral follicles (PFs) in sheep and buffalo. Small pre-antral follicles (SPFs, 100–250 μm) and large pre-antral follicles (LPFs, 250–450 μm) were isolated from slaughterhouse ovaries in the breeding seasons by a mechanical and enzymatic method. The sheep and buffalo PFs were cultured in vitro for 6 and 15 days, respectively, and examined for their growth, survival and antrum formation rates and growth rates of oocytes in cultured pre-antral follicles. The follicles of the sheep and buffalo were recovered and cultured simultaneously within replicates. The recovery rates (number per ovary) of both SPFs and LPFs were significantly (p < 0.05) higher in cyclic ewes (SPFs: 22.0 ± 3.3 vs 12.1 ± 2.6 and LPFs: 16.0 ± 3.6 vs 9.2 ± 1.8) and buffaloes (SPFs: 9.2 ± 1.3 vs 4.1 ± 1.0 and LPFs: 10.3 ± 2.7 vs 5.4 ± 0.7) compared with those recovered from acyclic ones. Presence of a DF in ovary significantly (p < 0.05) reduced the recovery rates of LPFs in ewes (9.06 ± 2.7 vs 16.4 ± 3.8) but had no effect in buffalo. Cyclicity of animals or follicular dominance had no effects on in vitro growth, survival and antrum formation rates and growth rates of oocytes in cultured PFs of SPFs and LPFs in both sheep and buffalo. The in vitro growth, survival and antrum formation rates of LPFs and growth rates of oocytes in cultured LPFs were significantly (p < 0.05) higher than those observed in SPFs in both sheep and buffalo. The overall recovery and growth rates of the PFs were lower in buffaloes compared with ewes.     

Purification of secretory immunoglobulin A from milk of the brushtail possum (Trichosurus vulpecula)

AIM: To identify and purify secretory immunoglobulin A (sIgA), a key effecter molecule in mucosal immune responses, from milk of the brushtail possum (Trichosurus vulpecula).

METHODS: Milk samples were collected from female possums with pouch young, and clarified by centrifugation and precipitation methods. The clarified fraction was purified by gel filtration and affinity chromatography to yield sIgA. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting techniques were used to assess the purity of the final product, and to identify the heavy (H) chain, light (L) chain and secretory component (SC) of possum sIgA.

RESULTS: Immunoblotting, using antibodies raised against cloned possum sIgA SC and H-chain, and a synthetic peptide fragment of the H-chain, confirmed the identity of the purified protein. The N-terminal amino acid sequence of purified possum sIgA showed strong homology to reported sequences of H-chain variable regions of marsupial immunoglobulins.

CONCLUSIONS: Milk was shown to be a convenient source of mucosal secretion containing sIgA, and a process involving 2 precipitation and 2 chromatography steps produced purified sIgA. This IgA preparation will prove useful for the generation of sIgA-specific immunological reagents for measurement of immune responses in the development of mucosal-based vaccines for biological control of possums.     

Supplementation with sunflower seeds in beef cattle did not impact on oocyte and in vitro embryo production

Supplementation with compounds rich in linoleic acid, including sunflower seed supplementation, promotes increase in conception rates in cows. We aimed to evaluate whether the sunflower seed (linoleic acid source) supplementation in beef donor females alters the plasma concentrations of cholesterol, triglycerides, HDL and LDL, increases the number and quality of oocytes, increases the cleavage rates and determines an improvement in number and quality of in vitro produced blastocysts. Thus, Nelore females were divided into two groups of 15 animals to receive supplementation with or without sunflower seed for 57 days. Females underwent follicular aspiration and the oocytes were subjected to in vitro embryo production. There was no difference (p > .1) between control group and group supplemented with sunflower seed on the number of displayed follicles; number of aspired oocytes; recovery rate; cleavage rate; number of embryos; number of blastocysts; embryos number of grades I and II; plasma concentrations of total cholesterol, triglycerides; HDL and LDL. Therefore, sunflower seed supplementation in oocyte donors did not increase the number and quality of oocytes, cleavage rates and the number and quality of blastocysts produced in vitro.     

Cellular Composition and Viability of Cloned Bovine Embryos Using Exogene-Transfected Somatic Cells

The present study compared the efficiency of transgenic (TG) cloned embryo production by somatic cell nuclear transfer (SCNT) with fetal-derived fibroblast cells (FFCs) which were transfected with pEGFP-N1 to in vitro-fertilized (IVF), parthenogenetic and SCNT counterparts by evaluating the rates of cleavage and blastocyst formation, apoptosis rate at different developmental stages, cell number, ploidy and gene expression in blastocysts. In SCNT and TG embryos, the rates of cleavage and blastocyst formation were significantly lower (p < 0.05) than those of IVF controls, but it did not differ between SCNT and TG embryos. In IVF control, 86.7% embryos displayed diploid chromosomal complements and the rates were significantly (p < 0.05) higher than those of SCNT and TG embryos. Most TG embryos (79%) with FFCs expressed the gene by both PCR and under fluorescence microscopy. The expression of apoptosis by TUNEL was first detected at six to eight cell stages in all embryos of IVF, SCNT and TG groups, but the expression rate at each developmental stages was significantly higher (p < 0.05) in SCNT and TG embryos than in IVF counterparts. The expression rate in inner cell mass (ICM) of TG embryos was significantly higher (p < 0.05) than in SCNT and IVF embryos. These results indicate that the high occurrence of apoptosis observed in SCNT and TG embryos compared with IVF counterparts might influence the developmental competence. Moreover, the SCNT embryos derived using non-transfected donor cells exhibited a lower apoptosis expression in ICM cells than in TG embryos derived using pEGP-N1-transfected donor cells suggesting a possible role of negative gene effect in TG embryos.     

Ultrasonographic Characterization of Follicle Deviation in Follicular Waves with Single Dominant and Codominant Follicles in Dromedary Camels (Camelus dromedarius)

Follicular wave emergence was synchronized by treating camels with GnRH when a dominant follicle (DF) was present in the ovaries. Animals were scanned twice a day from day 0 (day of GnRH treatment) to day 10, to characterize emergence and deviation of follicles during the development of the follicular wave. Follicle deviation in individual animals was determined by graphical method. Single DFs were found in 16, double DFs in 9 and triple DFs in two camels. The incidence of codominant (double and triple DFs) follicles was 41%. The interval from GnRH treatment to wave emergence, wave emergence to deviation, diameter and growth rate of F1 follicle before or after deviation did not differ between the animals with single and double DFs. The size difference between future DF(s) and the largest subordinate follicle (SF) was apparent from the day of wave emergence in single and double DFs. Overall, interval from GnRH treatment to wave emergence and wave emergence to the beginning of follicle deviation was 70.6 ± 1.4 and 58.6 ± 2.7 h, respectively. Mean size of the DF and largest SF at the beginning of deviation was 7.4 ± 0.2 and 6.3 ± 0.1 mm, respectively. In conclusion, the characteristics of follicle deviation are similar between the animals that developed single or double DFs.     

Husbandry Factors and the Resumption of Luteal Activity in Open and Zero‐Grazed Dairy Cows in Urban and Peri‐Urban Kampala,Uganda

The study investigated the influence of selected husbandry factors on interval to resumption of post‐partum cyclicity among dairy cows in urban and peri‐urban Kampala. A prospective study of 85 day post‐partum period of 59 dairy cows in open (n = 38) and zero grazing (n = 21) systems was conducted on 24 farms. Cows of parity 1–6 were recruited starting 15–30 days post‐partum. Progesterone (P4) content in milk taken at 10–12 day intervals was analysed using ELISA. The cow P4 profiles were classified into ‘normal’ (< 56 days), ‘delayed’ (> 56 days), ‘ceased’ or ‘prolonged’ (if started < 56 days but with abnormal P4 displays) resumption of luteal activity and tested for association with husbandry and cow factors. Of the 59 cows, luteal activity in 81.4% resumed normally and in 18.6%, delayed. Only 23.7% maintained regular luteal activity, while the others had ceased (10.2%), prolonged (37.3%) or unclear luteal activity (20.3%). There were no differences between open and zero‐grazed cows. Milk production was higher (p < 0.05) in zero than open grazing, in urban than peri‐urban and in cows fed on brew waste (p < 0.001) compared with mill products and banana peels. Results suggest that luteal activity resumes normally in a majority of cows, although only a minority experienced continued normal cyclicity once ovulation had occurred, in the two farming systems irrespective of feed supplements or water, and that supplementing with brew waste is beneficial for milk production.     

Dermatitis associated with microfilariae (Filarioidea) in 10 dogs

Dermatitis associated with microfilariae of a nematode of the superfamily Filarioidea was diagnosed in 10 dogs from the western United States. Clinically, lesions were single or multiple papules and plaques with alopecia, scarring, erythema, ulceration, or crusting. Eight dogs had lesional pruritus. Microscopically, there was perivascular, periglandular, to interstitial inflammation, with many eosinophils and/or plasma cells, and scarring affecting the dermis and subcutis. Microgranulomas containing microfilaria were seen in six dogs. Microfilariae were noted in microgranulomas or free in the dermis or subcutis, but not in vessels. In one case, an adult female nematode emerged from a biopsy sample that was placed in physiologic saline. Study of this nematode revealed that it was a filarioid of the family Onchocercidae; it was identified as Acanthocheilonema sp. (syn: Dipetalonema ). Antigen tests of five dogs were negative for Dirofilaria immitis . The Knott tests and/or filter tests of nine dogs were negative for microfilariae. An indirect fluorescence antibody test of one dog was also negative for D. immitis . One dog was not evaluated for microfilariae.     

Evaluation of vectorelectrocardiographic analysis of young broiler chickens as a predictive index for susceptibility to ascites syndrome.

Vectorelectrocardiographic (VCG) analysis was performed on 50 male broiler chicks (1 week of age) before placement in a hypobaric chamber. During 5 weeks of exposure to hypobaric hypoxia (simulated altitude of 2900 m), all recorded mortality (38%) was due to the development of ascites syndrome. There was a positive correlation (r = 0.74, P less than 0.01) between the increment in the frontal plane mean resultant vector magnitude divided by body weight (designated as the cardiac index [CI]), with the severity of right ventricular enlargement. Chicks developing ascites syndrome had a greater CI (P less than 0.05) at 1 week of age when compared with chicks that did not develop the syndrome. Therefore, the CI calculated by VCG analysis recognizes right ventricular enlargement, suggesting that a pre-hatch or early post-hatch functional cardiac stress has occurred, predisposing the 1-week-old broiler chick to ascites syndrome. Application of the CI has a physiological index may prove useful in future studies targeted for selection of ascites syndrome resistance in broiler chickens.     

Effect of used litter from floor pens of adult broilers on Salmonella colonization of broiler chicks.

The effect of used pine-shaving litter from broiler floor pens on Salmonella colonization resistance was evaluated in broiler chicks. One-day-old chicks were placed in floor pens on fresh unused litter or on used litter. All chicks were challenged orally with 10(4) S. typhimurium at 3 days of age. The study was replicated in three trials with used litter that was collected and stored for 1 day (Trial 1), 4 days (Trial 2), or 50 days (Trial 3) before the start of each trial. Cecal concentrations of volatile fatty acids (VFAs) were significantly higher (P < 0.05) in chicks placed on used litter than in chicks on new litter. In all three trials, the number of Salmonella in the cecal contents and the number of Salmonella cecal-culture-positive chicks was significantly lower (P < 0.01) at 10 days and 20 days of age in the chicks on used litter than in the chicks on new litter. The results indicated that newly hatched chicks reared on used litter had higher cecal VFA concentrations and higher resistance to Salmonella colonization than chicks reared on new litter.