The development and integrity of equine pre‐antral follicles cultured in vitro with follicle‐stimulating hormone (FSH) supplementation

This study investigated the effects of different concentrations of FSH (10, 50, 100 and 200 ng/ml) in supplemented MEM+ on the development of equine pre‐antral follicles that were cultured in vitro for 2 or 6 days. The ovaries (n = 5) from mares in seasonal anoestrus were collected from a local abattoir. Ten ovarian tissue fragments of approximately 3 × 3 × 1 mm were obtained from each animal. The fragments were cultured in situ for 2 days (D2) or 6 days (D6) in MEM+ or MEM+ supplemented with FSH at four different concentrations, establishing the following 11 groups: control (D0); MEM + (D2); MEM + (D6); MEM + 10 ng/ml of FSH (D2); MEM + 10 ng/ml of FSH (D6); MEM + 50 ng/ml of FSH (D2); MEM + 50 ng/ml of FSH (D6); MEM + 100 ng/ml of FSH (D2); MEM + 100 ng/ml of FSH (D6); MEM + 200 ng/ml of FSH (D2); and MEM + 200 ng/ml of FSH (D6). Follicles were observed in only 9.65% (388 of 4,018) of the histological sections. Of the 861 follicles evaluated, 488 were in the primordial stage, and 373 were in various developmental stages; 59.7% were morphologically normal. Regarding the integrity of the pre‐antral follicles, the groups with 100 ng/ml FSH of 2‐days culture as well as 50, 100 and 200 ng/ml FSH of 6‐days culture provided the best results. In conclusion, the in vitro culture of abattoir‐derived equine ovarian fragments presented better morphological integrity when supplemented with FSH for 6 days, in comparison with the MEM culture group. However, no clear effects were observed with FSH regarding the promotion of activation from a primordial to a developing follicle.     

Use of a Piezo Drill for Intracytoplasmic Sperm Injection into Cattle Oocytes Activated with Ionomycin Associated with Roscovitine

Intracytoplasmic sperm injection (ICSI) consists of the introduction, by micromanipulation, of a single sperm into the cytoplasm of a mature egg. This technique is particularly advantageous when only a few sperm are available for fertilization, representing an important tool in preserving genetic material, especially from poorly fertile males. The results from ICSI in cattle are very often unsatisfactory and difficult to reproduce. Thus, the goal of this study was to evaluate the effect of the use of a Piezo drill (PD) and oocyte activation with ionomycin + roscovitine (I + R) during ICSI in cattle oocytes. After in vitro maturation (24 h), cumulus complex oocytes were divided into four groups: G1 – the ICSI was performed without the use of a PD and the oocyte was activated with I + R; G2 – the ICSI was performed with the use of the PD and activation with I + R; G3 – the ICSI was performed with the use of the PD, but without activation and G4 – parthenogenetic control, treated with I + R, but without sperm injection. The presumptive zygotes were cultured for 7 days and evaluated on day 3 for cleavage rate and on day 7 for blastocyst formation. Embryo production by standard in vitro fertilization in the laboratory was 78% for cleavage (117/150) and 35% for blastocyst formation (41/150). The cleavage rates obtained in G1, G2 and G4 were similar (66.7%, 71.6% and 66.3%, respectively), demonstrating the beneficial effect of oocyte activation. However, in G3, despite the presence of the sperm and the electric stimulation of a PD, the cleavage rates were significantly lower (17.5%) compared with the groups that used chemical activation, even in the absence of sperm (G4). Despite the beneficial effects of activation, this stimulus alone, or in the absence of the PD, was not sufficient for adequate morulae formation (13.4%, 37.9%, 0.0% and 13.5% for G1, G2, G3 and G4, respectively). Only in G2, when the PD was used followed by artificial activation, blastocysts were obtained (14.7%). These results indicate that cattle oocytes must be activated after ICSI to produce viable embryos.     

Breed influences on in vitro development of abattoir-derived bovine oocytes

Background

There is a discrepancy in the reproductive performance between different cattle breeds. Using abattoir-derived ovaries and data base information we studied the effects of breed on in vitro fertilization and early embryo development.

Methods

The in vitro developmental competence of oocytes from cattle (n = 202) of Swedish Red (SR), Swedish Holstein (SH) and mixed beef breeds was compared, retrospectively tracing donors of abattoir-derived ovaries using a combination of the national animal databases and abattoir information. Age was significantly lower and carcass conformation score was higher in the beef breeds than in the dairy breeds.Cumulus oocyte complexes (n = 1351) were aspirated from abattoir-derived ovaries from animals of known breed (visual inspection confirmed through databases), age (databases), and abattoir information. Oocytes were matured, fertilized (frozen semen from two dairy bulls) and cultured according to conventional protocols. On day 8, blastocysts were graded and the number of nuclei determined.

Results

Cleavage rate was not different between the breeds but was significantly different between bulls. The percentage of blastocysts on day 8 was significantly higher when the oocyte donor’s breed was beef or SR than SH. There was no significant difference in blastocyst grades or stages between the breeds, but the number of nuclei in day 8 blastocysts was significantly lower in SH compared to the beef.

Conclusions

The use of abattoir-derived ovaries from animals whose background is traceable can be a valuable tool for research. Using this approach in the present study, oocyte donor breed was seen to affect early embryo development during in vitro embryo production, which may be a contributing factor to the declining fertility in some dairy breeds seen today.     

Antimicrobial susceptibility of bacteria isolated from neonatal foal samples submitted to a New Zealand veterinary pathology laboratory (2004 to 2013)

AIMS: To describe antimicrobial susceptibility, and identify antimicrobial resistance (AMR), in bacteria isolated from New Zealand foals.

METHODS: A database search was performed of submissions to a veterinary pathology laboratory between April 2004 and December 2013 for bacterial culture of samples from foals <3 weeks of age. Culture and susceptibility results were compiled with demographic information. Susceptibility results were as defined for the Kirby-Bauer disk diffusion susceptibility test based on Clinical Laboratory Standards Institute guidelines. Multi-drug resistance (MDR) was defined as non-susceptibility to ≥3 of a panel of antimicrobials (ceftiofur, enrofloxaxin, gentamicin, penicillin, tetracycline, trimethoprim-sulfonamide); penicillin susceptibility was not included for Gram-negative isolates.

RESULTS: Submissions from 102 foals were examined, and 127 bacterial isolates were cultured from 64 (63%) foals. Of the 127 isolates, 32 (25%) were Streptococcus spp., 30 (24%) were Staphylococcus spp., 12 (10%) were Enterococcus spp. and 26 (21%) were Escherichia coli. Of 83 Gram-positive isolates, 57 (69%) were susceptible to penicillin. Over all isolates, 92/126 (73%) were susceptible to gentamicin and 117/126 (93%) to enrofloxacin; 62/82 (76%) of Gram-positive, and 22/42 (52%) of Gram-negative bacteria were susceptible to ceftiofur; 53/81 (65%) of Gram-positive, and 23/44 (52%) of Gram-negative bacteria were susceptible to tetracycline; 59/82 (72%) of Gram-positive, and 23/44 (43%) of Gram-negative bacteria were susceptible to trimethoprim-sulfonamide. Of 126 isolates, 33 (26%) had MDR; >1 isolate with MDR was cultured from 24/64 (38%) foals, and ≥2 isolates with MDR were recovered from 8/64 (13%) foals.

CONCLUSIONS: Multi-drug resistance, including resistance to commonly used antimicrobials, was found in bacterial isolates from foals in New Zealand.

CLINICAL RELEVANCE: The results of this study are of concern from a treatment perspective as they indicate a potential for antimicrobial treatment failure. For future surveillance of AMR and the creation of national guidelines, it is important to record more data on samples submitted for bacterial culture.