Study of the Viability of Oocytes Collected from Bovine Ovaries Obtained in a Slaughterhouse Under Punctured at Different Times

Study Design

The bovine ovaries enrolled in this study were collected at the slaughterhouse “Beira Serra”, located at Oliveira do Hospital, Portugal. The collection was performed during 3 weeks (once a week). The biological material, 44 ovaries, was obtained from female bovines (crossbred beef cattle) aged between 11 and 17 months, selected for slaughter; and corresponded to material that was going to be discarded. The median age of the animals considering the number of ovaries obtained for each age category was 13 months. Of these ovaries, a total of 369 oocytes were punctured but for the study in Table 1. 164 that were puncture in the laboratory that exists in the slaughterhouse, and 205 that were punctured at the laboratory reproduction of the Faculty of Health Sciences, University of Beira Interior, Covilhã, Portugal.

The slaughter lasted about 4 hours. At the slaughter line, the ovaries were macroscopically assessed for the presence of follicles, which was the single criteria to be considered for this study. At the slaughter line, the ovaries were macroscopically evaluated for the presence of follicles and CL, to equitably distribute the ovaries by group in relation to the presence of CL.

When both ovaries from an individual were considered able to enter the study, one of its ovaries was punctured immediately after the end of slaughter, obtaining the oocytes that would be transported to the laboratory and the other was readied for transport for the laboratory where they were punctured (after 4 6 hours had passed, comprising time for puncture of ovaries at the slaughterhouse and transfer). After puncture, the oocytes were selected and matured, following the same procedure for both sets of oocytes (Figure 1).

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Puncture of bovine ovaries for oocyte collection at the slaughterhouse: The ovaries collected from cows at the slaughterhouse to be punctured in place, were immediately submerged in a Phosphate Buffer Saline 1x solution [PBS: 1.37M NaCl (Fisher Scientific, United States of America, USA), 27mM KCl (ChemLab, Belgium), 100mM Na2HPO4 (Fisher Scientific, New Hampshire, USA), 20mM KH2PO4 (ChemLab, Belgium)], supplemented with antibiotics (penicillin 100 IU/ml; streptomycin 100 µL/mL; Sigma-Aldrich, USA) and kept in a thermos bottle at 34-35°C. The complex-cumulus- oocytes (COCs) were aspirated from 3-8 mm follicles, with a 18G needle (Braun, Germany) attached to a 10 mL syringe (Terumo, Japan) and placed in a 50 mL falcon tube (Labbox, Spain) with oocyte collection medium. After collection, the fluid was poured into a 15 mL falcon tube (Labbox, Spain) containing maturation medium and transported to the laboratory in a thermos with water at 32ºC at room temperature (25-30°C). Upon arrival at the laboratory, 2 hours after puncture, the contents of the falcon tube were placed in a petri dish (90mm, VWR, USA). Puncture of bovine ovaries for oocyte collection at the laboratory: Those ovaries which were not processed immediately were collected from the same cows at the slaughterhouse and transported to the laboratory in a thermos at 32ºC at room temperature (25-30°C) in a container with PBS 1x solution supplemented with antibiotics. Once in the laboratory, the ovaries were washed in with PBS and kept in a water bath (J.P. Selecta, Spain) at 34-352°C. The puncture of ovaries and oocyte collection was performed as above described.

Bovine in Vitro Maturation of Oocytes (bIVM)

Culture media and solutions: The solutions used were the following: PBS solution 1x with antibiotics; collection medium: TCM 199 (Biochrom AG, United Kingdom), supplemented with HEPES (25mM, Fisher Scientific, USA), polyvinilic alcohol (PVA) (1mg/mL, Sigma-Aldrich, USA), antibiotics (penicilin 100 IU/mL; streptomycin 100 µL/mL, Sigma-Aldrich, USA), heparin (10 μg/mL, Sigma-Aldrich, USA) and glutamine (0.1 mg/mL, Alfa Aesar, USA); maturation medium: TCM 199, supplemented with Epidermal Growth Factor (10ng/ml, EGF, Gibco InVitrogen, USA), FSH/ LH (0.05 IU, Menopur, Ferring, USA), glutamine (0.1 mg/mL), sodium pyruvate (0.11 mg/ml, Sigma-Aldrich, USA) and PVA (1 mg/ mL). Experimental procedure: The experimental procedure used was the one described in ECVAM DB-ALM Protocol nº 129 -Toxicity test on in vitro maturation of bovine oocytes. Selection of oocytes and maturation: By using a stereo microscope (SZ, Olympus, Europe), grade I, II and III oocytes were selected, washed twice in collection medium, and transferred to a 4-wells tissue culture plate (VWR, USA) containing maturation medium (maximum 20 oocytes per well). This selection took place 8 hours after slaughter, and the time spent per group (oocytes punctured in the slaughterhouse and punctured in the laboratory) was about 30 minutes. The 4-wells plates were incubated at 5% CO2, 38.5°C, and saturated humidity for 24h in an incubator (UniTherm, CO2 Series, Uniequip GmbH, Germany). Oocyte denudation and microscopic examination: At the end of the incubation time, the granulosa and most of the cumulus oocytes cells were enzymatically removed, leaving only the corona radiata. For this procedure the oocytes of each well were collected and placed in contact with 100 µL of cumulase (Cumulase, Origio®, Denmark), for 1 minute and 20 seconds and then washed 5 times with maturation medium. The oocytes, after being stripped and placed in maturation medium, were observed under an inverted microscope (IX51, Olympus, Europe) under 400x magnification. Evaluation of oocytes maturation: The presence of a smaller, more compact refringent mass accompanying the secondary oocyte chromosomes corresponding to the extrusion of the first polar body (PB) associated to the second metaphase plate, was indicative of complete maturation. The number of oocytes in which PB extrusion was observed, and those in which it was not observed were registered. Oocytes in the germinal vesicle (GV), chromatin condensation (CC I and CC II) or germ vesicle disintegration (GVBD), metaphase I, anaphase I and telophase I stages were classified as non- matured [11].

The results were expressed as frequency of maturation (fm, %), considering the number of matured oocytes (mo) in Day Age (months) Animals (n) Frequency of matured oocytes (%; n) p-value Puncture at slaughterhouse Puncture at laboratory $$ 1 < = 1 3 \mathrm {m} 8 8 4 (2 7 / 3 2) 8 0 (2 8 / 3 5) \mathrm {N S} $$

1 >13m 7 87 (20/23) 77 (23/30) NS

$$ 2 < = 1 3 \mathrm {m} 9 9 5 (1 9 / 2 0) 8 5 (2 2 / 2 6) 0, 0 3 1 7 $$

2 >13m 9 100 (21/21) 94 (30/32) 0,0289

$$ 3 < = 1 3 \mathrm {m} 6 7 9 (3 3 / 4 2) 8 3 (3 5 / 4 2) \mathrm {N S} $$

3 >13m 5 85 (22/26) 90 (36/40) 0,0078

MEAN ± SD

$$ 8 8 \pm 8 \quad 8 5 \pm 6 \quad N S $$

NS: non-significant (p>0.05); SD: standard deviation. Table 2: Frequency of matured oocytes, after puncture at the slaughterhouse or at the laboratory. Number of matured oocytes in the total of punctured oocytes is also showed. The corresponding p-value highlighting statistical significance is showed, as well as data obtained for each group of animals.

We also analysed the results regarding the age of the animal at time of slaughter in Figure 2. Previous studies Morin L [12] have indicated that the age of the animal at time of puncture could influence the results. Specifically, oocytes the total of punctured oocytes (po):

Statistical Analysis

The differences between proportions (frequencies of maturation) were assessed for their statistical significance with the two-sided Chi-square test, using GraphPad prism v7.03 software (GraphPad Software, San Diego, California, USA). Results were statistically significant when p-value <0.05.